Poly-gamma-glutamic conjugates for eliciting immune responses directed against bacilli

ABSTRACT

Immunogenic compositions and methods for eliciting an immune response against  B. anthracis  and other bacilli are provided that include immunogenic conjugates of a poly-γ-glutamic acid (γPGA) polypeptide of  B. anthracis,  or of another  Bacillus  that expresses a γPGA polypeptide. The γPGA conjugates elicit an effective immune response against  B. anthracis,  or against another  Bacillus,  in mammalian hosts to which the conjugates are administered.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional PatentApplication No. 60/476,59 filed Jun. 5, 2003, which is incorporatedherein by reference in its entirety.

FIELD OF THE DISCLOSURE

This invention relates to the field of immunology and, morespecifically, to immunogenic compositions and methods for eliciting aneffective immune response against Bacillus anthracis (. anthracis).

BACKGROUND

Anthrax is an acute infectious disease caused by the bacterium B.anthracis. Anthrax m

commonly occurs in wild and domestic lower vertebrates (cattle, sheep,goats, camels, antelopes, other herbivores), but it can also occur inhumans, for example, when they are exposed to infected animals or tissuefrom infected animals, or anthrax spores.

The virulence of B. anthracis is dependent on Anthrax Toxin (AT), andthe poly-γ-D-glutamic acid capsule (γDPGA). The genes for the toxin, andthe capsule, are carried by plasmids designated pX01 and pX02,respectively (Mikesell et al, Infect. Immun. 39:371-76, 1983; Vodkinal., Cell 34:693-97, 1983; Green et al., Infect. Immun. 49:291-97,1985). AT is composed of three entities: Protective Antigen (PA) (thebinding subunit of AT), and two enzymes known as Lethal Factor (LF) andEdema Factor (EF) (Mikesell et al., Infect. Immun. 39:371-76, 1983;Vodkin et al Cell 34:693-97, 1983). PA is an 83 kDa protein that is themain protective constituent of anthrax vaccines.

PA is necessary for vaccine immunogenicity (Ivins et al., Infect. Immun.60:662-68, 199

Welkos and Friedlander, Microb. Pathog. 5:127, 1998). Antibodies againstPA prevent anthrax to from binding to host cells, thus abrogatingtoxicity (Little and Ivins, Microbes. Infect. 1:131-39, 1999).Additionally, antibodies to PA can inhibit the germination of sporeswhile improving their phagocytosis and killing by macrophages (Welkoseta., Microbiology 147:1677-85, 2001). Unfortunately, the currentlylicensed human anthrax vaccine (AVA, BioPort Corporation, Lansing Mich.)requires six vaccinations over eighteen months followed by yearlyboosters to induce and maintain protective anti-PA titers (Pittman etal., Vaccine 20:1412-20, 2002; Pittman et al., Vaccine 20:972-78, 2001).In some vaccines, this regimen is associated with undesirable localreactogenic (Pittman et al., Vaccine 20:972-78, 2001).

Thus, while certain prophylactic and treatment schemes may prove usefulin preventing o directed toward anthrax. In particular, there is a needfor an effective and safe vaccine that would require fewer doses toconfer immunity to anthrax.

BRIEF SUMMARY OF SPECIFIC EMBODIMENTS

An immunogenic conjugate is disclosed herein The immunogenic conjugateincludes a Bacillus capsular poly-γ-glutamic acid (γPGA) polypeptidecovalently linked to a carrier, wherein the conjugate elicits an immuneresponse in a subject. A composition including the immunogenic conjugateand a pharmaceutically acceptable carrier is also disclosed herein.

A method of eliciting an immune response against a Bacillus antigenicepitope in a subject is also disclosed. The method includes introducinginto the subject a composition including the immunogenic conjugate and apharmaceutically acceptable carrier, thereby eliciting an immuneresponse in the subject. Optionally, the composition includes anadjuvant.

Further disclosed herein are isolated antibodies that bind to theBacillus γPGA polypeptide. In one embodiment, the isolated antibodiesrecognize antigenic epitopes on both the Bacillus γPGA polypeptide andthe carrier protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a GLC-MS spectrum analysis of the rPA-Cys-Gly₃-γPGA₁₀-Cconjugate, demonstrating that L-Glu can be separated from D-Glu andmeasured in order to calculate the number of γDPGA chains incorporatedinto the protein of the conjugate.

FIG. 2A-2D are a set of MALDI-TOF spectra, showing the mass spectra ofrecombinant B. anthracis rPA (FIG. 2A); Br-rPA (FIG. 2B);rPA-Cys-Gly₃-γDPGA₁₀-C conjugate containing an average of 11 γDPGAchains per rPA (FIG. 2C); and rPA-Cys-Gly₃-γDPGA₁₀-C conjugatecontaining an average of 16 γDPGA chains per rPA (FIG. 2D).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 is the amino acid sequence of human immunodeficiency virus(HIV)-1 Tat protein.

SEQ ID NOs: 2 and 3 show the nucleic and amino acid sequences of B.anthracis protective antigen.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

I. Abbreviations

ADH: adipic acid dihydrazide

AT: anthrax toxin

ATR: anthrax toxin receptor

EDAC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl

EF: edema factor

γPGA: poly-γ-glutamic acid capsule from a Bacillus

γDPGA: poly-γ-D-glutamic acid capsule from B. anthracis

γLPGA: poly-γ-L-glutamic acid capsule from a Bacillus

GLC-MS: gas-liquid chromatography-mass spectrometry

kDa: kilodaltons

LC-MS: liquid chromatography-mass spectrometry

LeTx: lethal toxin

LF: lethal factor

LPS: lipopolysaccharide

MALDI-TOF: matrix-assisted laser desorption ionization time-of-flight

μg: microgram

μl: microliter

PA: protective antigen

PBS: phosphate buffered saline

rEPA: recombinant Pseudomonas aeruginosa exotoxin A

rPA: recombinant B. anthracis protective antigen

SBAP: succinimidyl 3-(bromoacetamido)propionate

SFB: succinimidylformylbenzoate

SPDP: N-hydroxysuccinimide ester of 3-(2-pyridyldithio)propionic acid

SLV: succinimidyllevulinate

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Publishers, 1994 (ISBN0632021829); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. Also, as used herein, the term “comprises” means“includes.” Hence “comprising A or B” means including A, B, or A and B.It is further to be understood that all nucleotide sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent disclosure, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Adjuvant: A substance that non-specifically enhances the immune responseto an antigen. Development of vaccine adjuvants for use in humans isreviewed in Singh et al. (Nat. Biotechnol. 17:1075-1081, 1999), whichdiscloses that, at the time of its publication, aluminum salts, such asaluminum hydroxide (Amphogel, Wyeth Laboratories, Madison, N.J.), andthe MF59 microemulsion are the only vaccine adjuvants approved for humanuse.

In one embodiment, an adjuvant includes a DNA motif that stimulatesimmune activation, for example the innate immune response or theadaptive immune response by T-cells, B-cells, monocytes, dendriticcells, and natural killer cells. Specific, non-limiting examples of aDNA motif that stimulates immune activation include CpGoligodeoxynucleotides, as described in U.S. Pat. Nos. 6,194,388;6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and6,429,199.

Analog, Derivative or Mimetic: An analog is a molecule that differs inchemical structure from a parent compound, for example a homolog(differing by an increment in the chemical structure, such as adifference in the length of an allyl chain), a molecular fragment, astructure that differs by one or more functional groups, a change inionization. Structural analogs are often found using quantitativestructure activity relationships (QSAR), with techniques such as thosedisclosed in Remington (The Science and Practice of Pharmacology, 19thEdition (1995), chapter 28). A derivative is a biologically activemolecule derived from the base structure. A mimetic is a molecule thatmimics the activity of another molecule, such as a biologically activemolecule. Biologically active molecules can include chemical structuresthat mimic the biological activities of a compound.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects, for example, humans, non-human primates,dogs, cats, horses, and cows.

Antibody: A protein (or protein complex) that includes one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” (about 50-70 kDa) chain. The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and “variable heavy chain” (V_(H)) refer, respectively, to theselight and heavy chains.

As used herein, the term “antibodies” includes intact immunoglobulins aswell as a number of well-characterized fragments. For instance, Fabs,Fvs, and single-chain Fvs (SCFvs) that bind to target protein (orepitope within a protein or fusion protein) would also be specificbinding agents for that protein (or epitope). These antibody fragmentsare defined as follows: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain; (2) Fab′, the fragment ofan antibody molecule obtained by treating whole antibody with pepsin,followed by reduction, to yield an intact light chain and a portion ofthe heavy chain; two Fab′ fragments are obtained per antibody molecule;(3) (Fab′)₂, the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)₂, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody, a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule. Methods of makingthese fragments are routine (see, for example, Harlow and Lane, UsingAntibodies: A Laboratory Manual, CSHL, New York, 1999).

Antibodies for use in the methods and devices of this disclosure can bemonoclonal or polyclonal. Merely by way of example, monoclonalantibodies can be prepared from murine hybridomas according to theclassical method of Kohler and Milstein (Nature 256:495-97, 1975) orderivative methods thereof. Detailed procedures for monoclonal antibodyproduction are described in Harlow and Lane, Using Antibodies: ALaboratory Manual, CSHL, New York, 1999.

Antigen: A compound, composition, or substance that may be specificallybound by the products of specific humoral or cellular immunity, such asan antibody molecule or T-cell receptor. In one embodiment, an antigenis a Bacillus antigen, such as γPGA.

Bacillus: A genus of bacteria whose collective features includedegradation of most substrates derived from plant and animal sources,including cellulose, starch, pectin, proteins, agar, hydrocarbons, andothers; antibiotic production; nitrification; denitrification; nitrogenfixation; facultative lithotrophy; autotrophy; acidophily; alkaliphily;psychrophily, thermophily and parasitism. Spore formation, universallyfound in the genus, is thought to be a strategy for survival in the soilenvironment, wherein the bacteria predominate. Aerial distribution ofdormant spores likely explains the occurrence of Bacillus species inmost habitats examined.

There are more than 40 recognized species in the genus Bacillus(Bergey's Manual of Systematic Bacteriology Vol. 2 (1986)). Theseinclude, but are not limited to, B. acidocaldarius, B. alkalophilus, B.alvei, B. anthracis, B. azotoformans, B. badius, B. brevis, B. cereus,B. circulans, B. coagulans, B. fastidiosis, B. firmus, B. globisporus,B. insolitus, B. larvae, B. laterosporus, B. lentinorbus, B. lentus, B.licheniformis, B. macerans, B. macquarienis, B. marinus, B. megaterium,B. mycoides, B. pantothenticus, B. pasteurii, B. polymyxa, B. popillia,B. pumilus, B. schlegelii, B. sphaericus, B. stearothermophilus, B.subtilis, and B. thuringiensis. In one specific, non-limiting example, aBacillus is Bacillus anthracis, the agent that causes anthrax.

Bacillus Anthracis: The etiologic agent of anthrax, Bacillus anthracisis a large, gram-positive, nonmotile, spore-forming bacterial rod. Thevirulence of B. anthracis is dependent on AT, and the γDPGA capsule. Thegenes for the toxin, and the capsule, are carried by plasmids,designated pX01 and pX02, respectively (Mikesell et al., Infect Immune.39:371-76, 1983; Vodkin et al., Cell 34:693-97, 1983; Green et al.,Infect Immun. 49:291-97, 1985).

AT is composed of three entities: PA (the binding subunit of AT), andtwo enzymes known as LF and EF (Mikesell et al., Infect. Immun.39:371-76, 1983; Vodkin et al., Cell 34:693-97, 1983). PA is an 83 kDaprotein that is the main protective constituent of anthrax vaccines. PAbinds to the anthrax toxin receptor (ATR) on cells and is thenproteolytically cleaved by the enzyme furin with release of a 20 kDafragment (Bradley et al., Nature 414:225-29, 2001; Klimpel et al., PNAS89:10277-81, 1992). The 63 kDa PA remnant (PA₆₃) features a secondbinding domain and binds to either EF, an 89 kDa protein, to form edematoxin, or LF, a 90 kDa protein, to form lethal toxin (LeTx) (Leppla etal., Salisbury Med. Bull. Suppl. 68:41-43, 1990). The resulting complexis internalized into the cell within endosomes (Singh et al, Infect.Immun. 67:1853-59, 1999; Friedlander, J. Biol. Chem. 261:7123-26, 1986).

The γDPGA capsule of B. anthracis serves as an essential virulencefactor during anthrax infection, inhibiting host defense mechanismsthrough inhibition of phagocytosis of the vegetative cells bymacrophages. While other Bacillus produce γPGA in a mixture of both D-and L-forms, only B. anthracis is known to synthesize it exclusively ina D-conformation (Kovács et al., J Chem. Soc. 4255-59, 1952). Wheninjected, γDPGA has been shown to be a poor immunogen (Eisner, Schweiz.Z. Pathol. Bakteriol. 22:129-44, 1959; Ostroff et al., Proc. Soc. Exp.Biol. Med. 99:345-47, 1958). The capsule also shields the vegetativeform of B. anthracis from agglutination by monoclonal antibodies to itscell wall polysaccharide (Ezzell et al., J. Clin. Microbiol. 28:223-31,1990).

Carrier: An immunogenic macromolecule to which an antigenic but nothighly immunogenic molecule, such as, for example, a homopolymer ofγPGA, can be bound. When bound to a carrier, the bound molecule becomesmore immunogenic. Carriers are chosen to increase the immunogenicity ofthe bound molecule and/or to elicit antibodies against the carrier whichare diagnostically, analytically, and/or therapeutically beneficial.Covalent linking of a molecule to a carrier confers enhancedimmunogenicity and T-cell dependence (Pozsgay et al., PNAS 96:5194-97,1999; Lee et al, J. Immunol. 116:1711-18, 1976; Dintzis et al., PNAS73:3671-75, 1976). Useful carriers include polymeric carriers, which canbe natural (for example, polysaccharides, polypeptides or proteins frombacteria or viruses), semi-synthetic or synthetic materials containingone or more functional groups to which a reactant moiety can beattached.

Examples of bacterial products for use as carriers include bacterialtoxins, such as B. anthracis PA (including fragments that contain atleast one antigenic epitope and analogs or derivatives capable ofeliciting an immune response), LF and LeTx, and other bacterial toxinsand toxoids, such as tetanus toxin/toxoid, diphtheria toxin/toxoid, P.aeruginosa exotoxin/toxoid/, pertussis toxin/toxoid, and C. perfringensexotoxin/toxoid. Viral proteins, such as hepatitis B surface antigen andcore antigen can also be used as carriers, as well as proteins fromhigher organisms such as keyhole limpet hemocyanin, horseshoe crabhemocyanin, edestin, mammalian serum albumins, and mammalianimmunoglobulins. Additional bacterial products for use as carriersinclude bacterial wall proteins and other products (for example,streptococcal or staphylococcal cell walls and lipopolysaccharide(LPS)).

Covalent Bond: An interatomic bond between two atoms, characterized bythe sharing of one or more pairs of electrons by the atoms. The terms“covalently bound” or “covalently linked” refer to making two separatemolecules into one contiguous molecule. The terms include reference tojoining a γPGA polypeptide directly to a carrier molecule, and tojoining a γPGA polypeptide indirectly to a carrier molecule, with anintervening linker molecule.

Epitope: An antigenic determinant. These are particular chemical groupsor contiguous or non-contiguous peptide sequences on a molecule that areantigenic, that is, that elicit a specific immune response. An antibodybinds a particular antigenic epitope based on the three dimensionalstructure of the antibody and the matching (or cognate) epitope.

γPGA: A homopolymer of glutamic acid residues linked by γ peptide bonds.The glutamic acid residues constituting the γPGA homopolymer can besolely in the L-form (γLPGA) or the D-form (γDPGA). When the form of theglutamic acid residues constituting the γPGA homopolymer can be eitherthe L-form or the D-form, or when the two forms are mixed in a singlemolecule, the term γPGA is used. The weakly immunogenic andantiphagocytic capsule found on many species of Bacillus, whichdisguises the bacilli from immune surveillance, consists of γPGA.

γPGA Conjugate: A naturally occurring γPGA polypeptide produced by B.anthracis or another Bacillus species or strain covalently linked to acarrier, as well as conjugates of a carrier with a polypeptide fragment,synthetic polypeptide, or chemically modified derivative of a γPGApolypeptide. In some embodiments, the γPGA conjugate will comprise aconjugate of a carrier protein with a synthetic γPGA polypeptide havinga selected peptide length and corresponding to a portion of a γPGApolypeptide from B. anthracis or another Bacillus species or strain thatpossesses a γPGA capsule.

Homopolymer: This term refers to a polymer formed by the bondingtogether of multiple units of a single type of molecular species, suchas a single monomer (for example, an amino acid).

Immune Response: A response of a cell of the immune system, such as aB-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus. Animmune response can include any cell of the body involved in a hostdefense response for example, an epithelial cell that secretesinterferon or a cytokine. An immune response includes, but is notlimited to, an innate immune response or inflammation.

Immunogenic Conjugate or Composition: A term used herein to mean acomposition useful for stimulating or eliciting a specific immuneresponse (or immunogenic response) in a vertebrate. In some embodiments,the immunogenic response is protective or provides protective immunity,in that it enables the vertebrate animal to better resist infection ordisease progression from the organism against which the immunogeniccomposition is directed.

Without wishing to be bound by a specific theory, it is believed that animmunogenic response can arise from the generation of an antibodyspecific to one or more of the epitopes provided in the immunogeniccomposition. The response can include a T-helper or cytotoxic cell-basedresponse to one or more of the epitopes provided in the immunogeniccomposition. All three of these responses may originate from naive ormemory cells. A response can also include production of cytokines. Onespecific example of a type of immunogenic composition is a vaccine.

Immunogen: A compound, composition, or substance which is capable, underappropriate conditions, of stimulating the production of antibodies or aT-cell response in an animal, including compositions that are injectedor absorbed into an animal.

Immunologically Effective Dose: An immunologically effective dose of theγPGA conjugates of the disclosure is therapeutically effective and willprevent, treat, lessen, or attenuate the severity, extent or duration ofa disease or condition, for example, infection by B. anthracis.

Inhibiting or Treating a Disease: Inhibiting the fill development of adisease or condition, for example, in a subject who is at risk for adisease such as anthrax. “Treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition after it has begun to develop. As used herein,the term “ameliorating,” with reference to a disease, pathologicalcondition or symptom, refers to any observable beneficial effect of thetreatment. The beneficial effect can be evidenced, for example, by adelayed onset of clinical symptoms of the disease in a susceptiblesubject, a reduction in severity of some or all clinical symptoms of thedisease, a slower progression of the disease, a reduction in the numberof relapses of the disease, an improvement in the overall health orwell-being of the subject, or by other parameters well known in the artthat are specific to the particular disease.

Isolated: An “isolated” microorganism (such as a virus, bacterium,fungus, or protozoan) has been substantially separated or purified awayfrom microorganisms of different types, strains, or species.Microorganisms can be isolated by a variety of techniques, includingserial dilution and culturing.

An “isolated” biological component (such as a nucleic acid molecule,protein or organelle) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, such as other chromosomal andextra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell, as well as chemically synthesized nucleic acids orproteins, or fragments thereof.

Linker: A molecule that joins two other molecules, either covalently, orthrough ionic, van der Waals or hydrogen bonds.

Opsonin: A macromolecule that becomes attached to the surface of amicrobe and can be recognized by surface receptors of neutrophils andmacrophages and that increases the efficiency of phagocytosis of themicrobe. Opsonins include IgG antibodies, which are recognized by theFcγ receptor on phagocytes, and fragments of complement proteins, whichare recognized by CR1 (CD35) and by the leukocyte integrin Mac-1.

Opsonophagocytosis: The process of attaching opsonins to microbialsurfaces to target the microbes for phagocytosis.

PA-based Immunogen: A term used herein to refer to all forms of PA whichare useful in immunogenic compositions and/or methods of the disclosure,including unmodified native or recombinant B. anthracis PA, or a variantor fragment thereof. Variants and fragments of PA are effective toelicit an anti-PA immune response in a subject to whom they areadministered.

Pharmaceutically Acceptable Carriers: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th Edition (1975), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compounds or molecules, such as one or more SARS-CoV nucleicacid molecules, proteins or antibodies that bind these proteins, andadditional pharmaceutical agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used. The terms “polypeptide” or “protein” as used herein areintended to encompass any amino acid sequence and include modifiedsequences such as glycoproteins. The term “polypeptide” is specificallyintended to cover naturally occurring proteins, as well as those whichare recombinantly or synthetically produced.

The term “residue” or “amino acid residue” includes reference to anamino acid that is incorporated into a protein, polypeptide, or peptide.

Conservative amino acid substitutions are those substitutions that, whenmade, least interfere with the properties of the original protein, thatis, the structure and especially the function of the protein isconserved and not significantly changed by such substitutions. Examplesof conservative substitutions are shown below. Original ResidueConservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys SerGln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; GluMet Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe ValIle; Leu

Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

The substitutions which in general are expected to produce the greatestchanges in protein properties will be non-conservative, for instancechanges in which (a) a hydrophilic residue, for example, seryl orthreonyl, is substituted for (or by) a hydrophobic residue, for example,leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, for example, lysyl, arginyl, orhistadyl, is substituted for (or by) an electronegative residue, forexample, glutamyl or aspartyl; or (d) a residue having a bulky sidechain, for example, phenylalanine, is substituted for (or by) one nothaving a side chain, for example, glycine.

Protective Antigen (PA): One of the three components of the anthraxtoxin. PA is a secreted nontoxic protein with a molecular weight of 83kDa and is the major protective constituent of anthrax vaccines. PAbinds to the ATR on cells and is then proteolytically cleaved by theenzyme furin with release of a 20 kDa fragment (Bradley et al., Nature414:225-29, 2001; Klimpel et al., PNAS 89:10277-81, 1992). The 63 kDa PAremnant (PA₆₃) features a second binding domain and binds to either EF,an 89 kDa protein, to form edema toxin, or LF, a 90 kDa protein, to formlethal toxin (LeTx). The sequence of PA is known, for example, asencoded by bases 143779 to 146073 of GenBank Accession No. NC 007322(plasmid pXO1; SEQ ID NOs: 2 and 3, nucleic and amino acid sequences,respectively).

Protein: A biological molecule, particularly a polypeptide, expressed bya gene and comprised of amino acids.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purifiedpeptide, protein, γPGA conjugate, or other active compound is one thatis isolated in whole or in part from naturally associated proteins andother contaminants, wherein the peptide, protein, γPGA conjugate, orother active compound is purified to a measurable degree relative to itsnaturally occurring state, for example, relative to its purity within acell extract. In certain embodiments, the term “substantially purified”refers to a peptide, protein, γPGA conjugate, or other active compoundthat has been isolated from a cell, cell culture medium, or other crudepreparation and subjected to fractionation to remove various componentsof the initial preparation, such as proteins, cellular debris, and othercomponents. Such purified preparations can include materials in covalentassociation with the active agent, such as glycoside residues ormaterials admixed or conjugated with the active agent, which may bedesired to yield a modified derivative or analog of the active agent orproduce a combinatorial therapeutic formulation, conjugate, fusionprotein or the like. The term purified thus includes such desiredproducts as peptide and protein analogs or mimetics or otherbiologically active compounds wherein additional compounds or moietiesare bound to the active agent in order to allow for the attachment ofother compounds and/or provide for formulations useful in therapeutictreatment or diagnostic procedures. Generally, substantially purifiedpeptides, proteins, γPGA conjugates, or other active compounds for usewithin the disclosure comprise more than 80% of all macromolecularspecies present in a preparation prior to admixture or formulation ofthe peptide, protein, γPGA conjugate or other active compound with apharmaceutical carrier, excipient, buffer, absorption enhancing agent,stabilizer, preservative, adjuvant or other co-ingredient in a completepharmaceutical formulation for therapeutic administration. Moretypically, the peptide, protein, γPGA conjugate or other active compoundis purified to represent greater than 90%, often greater than 95% of allmacromolecular species present in a purified preparation prior toadmixture with other formulation ingredients. In other cases, thepurified preparation may be essentially homogeneous, wherein othermacromolecular species are not detectable by conventional techniques.

Recombinant Nucleic Acid: A sequence that is not naturally occurring orhas a sequence that is made by an artificial combination of twootherwise separated segments of sequence. This artificial combination isoften accomplished by chemical synthesis or, more commonly, by theartificial manipulation of isolated segments of nucleic acids, forexample, by genetic engineering techniques such as those described inSambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2^(nd)ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The term recombinant includes nucleic acids that have beenaltered solely by addition, substitution, or deletion of a portion ofthe nucleic acid.

Specific Binding Agent: An agent that binds substantially only to adefined target. Thus a protein-specific binding agent bindssubstantially only the defined protein, or to a specific region withinthe protein. As used herein, a specific binding agent includesantibodies and other agents that bind substantially to a specifiedpolypeptide. The antibodies may be monoclonal or polyclonal antibodiesthat are specific for the polypeptide, as well as immunologicallyeffective portions (“fragments”) thereof.

The determination that a particular agent binds substantially only to aspecific polypeptide may readily be made by using or adapting routineprocedures. One suitable in vitro assay makes use of the Westernblotting procedure (described in many standard texts, including Harlowand Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).

Spore: A small, usually single-celled reproductive body that is highlyresistant to desiccation and heat and is capable of growing into a neworganism, produced especially by certain bacteria, fungi, algae, andnon-flowering plants. Spores have proven to be the most durable type ofcell found in nature, and in their cryptobiotic state of dormancy, theycan remain viable for extremely long periods of time, perhaps millionsof years. Spores do not form normally during active growth and celldivision. Rather, their differentiation begins when a population ofvegetative cells passes out of the exponential phase of growth, usuallyas a result of nutrient depletion. Typically, one spore is formed pervegetative cell. In some examples, the mature spore is liberated bylysis of the mother cell (sporangium) in which it was formed.

Mature spores have no detectable metabolism, a state that is describedas cryptobiotic. They are highly resistant to environmental stressessuch as high temperature (some spores can be boiled for several hoursand retain their viability), irradiation, strong acids, disinfectants,and the like. Although cryptobiotic, they retain viability indefinitelysuch that under appropriate environmental conditions, they germinateinto vegetative cells.

Therapeutically Effective Amount: A quantity of a specified agentsufficient to achieve a desired effect in a subject being treated withthat agent. For example, this may be the amount of a γDPGA conjugateuseful in increasing resistance to, preventing, ameliorating, and/ortreating infection and disease caused by B. anthracis infection in asubject Ideally, a therapeutically effective amount of an agent is anamount sufficient to increase resistance to, prevent, ameliorate, and/ortreat infection and disease caused by B. anthracis infection in asubject without causing a substantial cytotoxic effect in the subject.The effective amount of an agent useful for increasing resistance to,preventing, ameliorating, and/or treating infection and disease causedby B. anthracis infection in a subject will be dependent on the subjectbeing treated, the severity of the affliction, and the manner ofadministration of the therapeutic composition.

Toxoid: A nontoxic derivative of a bacterial exotoxin produced, forexample, by formaldehyde or other chemical treatment. Toxoids are usefulin the formulation of immunogenic compositions because they retain mostof the antigenic properties of the toxins from which they were derived.

III. Description of Several Embodiments

A. Bacillus γPGA Polypeptide—Carrier Conjugates

Bacillus capsular γPGA polypeptide—carrier conjugates (γPGA conjugates),are disclosed herein. The γPGA conjugates elicit an immune response in asubject, and inhibit or treat infection and/or disease caused by B.anthracis or other bacilli.

The weakly immunogenic and antiphagocytic γPGA capsule, which consistsof glutamic acid residues linked by γ peptide bonds, disguises thebacilli from immune surveillance. As disclosed herein, Bacillus capsularγPGA polypeptides include, but are not limited to, B. anthracis, B.licheniformis, B. pumilus, and B. subtilis γPGA polypeptides. AllBacillus besides B. anthracis that are known to produce γPGA make amixture of both the D- and L-forms, whereas B. anthracis producesexclusively γDPGA. In one embodiment, the γPGA conjugates disclosedherein are γLPGA conjugates. In another embodiment, the γPGA conjugatesare γDPGA conjugates. In a specific, non-limiting example, the γDPGAconjugate is a B. anthracis γDPGA conjugate.

Bacillus capsular γPGA polypeptides can be isolated by many methods wellknown in the art, such as salt fractionation, phenol extraction,precipitation with organic solvents (for example,hexadecyltrimethylammonium bromide (cetavlon) or ethanol), affinitychromatography, ion-exchange chromatography, hydrophobic chromatography,high performance liquid chromatography, gel filtration, isoelectricfocusing, and the like. In one specific, non-limiting example, Bacilluscapsular γPGA polypeptides are extracted from the culture supernatant ofgrowing bacilli by cetavlon precipitation, acidification to pH 1.5,precipitation with ethanol, and passage through a 2.5×100 cm SepharoseCL-4B column in 0.2M NaCl. The compositions of extracted γPGApolypeptides are determined by methods well known in the art, such as¹H-nuclear magnetic resonance (NMR) spectroscopy and ¹³C-NMRspectroscopy; while their enantiomeric confirmations can be determinedby gas-liquid chromatography-mass spectrometry (GLC-MS).

Synthetic γPGA polypeptides of varying lengths (for example, about 5,10, 15, or 20 residues) having either the D- or L-configuration can bereadily synthesized by automated solid phase procedures well known inthe art Suitable syntheses can be performed by utilizing “T-boc” or“F-moc” procedures. Techniques and procedures for solid phase synthesisare described in Solid Phase Peptide Synthesis: A Practical Approach, byE. Atherton and R. C. Sheppard, published by IRL, Oxford UniversityPress, 1989. In specific, non-limiting examples, the synthetic γPGApolypeptide includes about 1 to about 20 glutamic acid residues, such asabout 10 to about 15 glutamic acid residues, or about 10 glutamic acidresidues. The compositions and purity of synthetic γPGA polypeptides canbe determined by GLC-MS and matrix-assisted laser desorption ionizationtime-of-flight (MALDI-TOF) spectrometry.

Carriers for linking to γPGA polypeptides as disclosed herein are chosento increase the immunogenicity of the γPGA polypeptides and/or to elicitantibodies against the carrier which are diagnostically, analytically,and/or therapeutically beneficial. Covalent linking of γPGA polypeptidesto a carrier confers enhanced immunogenicity and T-cell dependence.Useful carriers include polymeric carriers, which can be natural,semi-synthetic or synthetic materials containing one or more functionalgroups, for example primary and/or secondary amino groups, azido groups,hydroxyl groups, or carboxyl groups, to which a reactant moiety can beattached. The carrier can be water soluble or insoluble, and in someembodiments is a protein or polypeptide. Carriers that fulfill thesecriteria are generally known in the art (see, for example, Fattom etal., Infect. Immun. 58:2309-12, 1990; Devi et al., PNAS 88:7175-79,1991; Szu et al., Infect Immun. 59:4555-61, 1991; Szu et al., J. Exp.Med 166:1510-24, 1987; and Pavliakova et al., Infect. Immun. 68:2161-66,2000).

Specific, non-limiting examples of water soluble polypeptide carriersinclude, but are not limited to, natural, semi-synthetic or syntheticpolypeptides or proteins from bacteria or viruses. In one embodiment,bacterial products for use as carriers include bacterial wall proteinsand other products (for example, streptococcal or staphylococcal cellwalls and LPS), and soluble antigens of bacteria. In another embodiment,bacterial products for use as carriers include bacterial toxins.Bacterial toxins include bacterial products that mediate toxic effects,inflammatory responses, stress, shock, chronic sequelae, or mortality ina susceptible host. Specific, non-limiting examples of bacterial toxinsinclude, but are not limited to: B. anthracis PA (for example, asencoded by bases 143779 to 146073 of GenBank Accession No. NC 007322,herein incorporated by reference), including variants that share atleast 90%, at least 95%, or at least 98% amino acid sequence homology toPA, fragments that contain at least one antigenic epitope, and analogsor derivatives capable of eliciting an immune response; B. anthracis LF(for example, as encoded by the complement of bases 149357 to 151786 ofGenBank Accession No. NC 007322); bacterial toxins and toxoids, such astetanus toxin/toxoid (for example, as described in U.S. Pat. Nos.5,601,826 and 6,696,065); diphtheria toxin/toxoid (for example, asdescribed in U.S. Pat. Nos. 4,709,017 and 6,696,065); P. aeruginosaexotoxin/toxoid/ (for example, as described in U.S. Pat. Nos. 4,428,931,4,488,991 and 5,602,095); pertussis toxin/toxoid (for example, asdescribed in U.S. Pat. Nos. 4,997,915, 6,399,076 and 6,696,065); and C.perfringens exotoxin/toxoid (for example, as described in U.S. Pat. Nos.5,817,317 and 6,403,094). Viral proteins, such as hepatitis B surfaceantigen (for example, as described in U.S. Pat. Nos. 5,151,023 and6,013,264) and core antigen (for example, as described in U.S. Pat. Nos.4,547,367 and 4,547,368) can also be used as carriers, as well asproteins from higher organisms such as keyhole limpet hemocyanin,horseshoe crab hemocyanin, edestin, mammalian serum albumins, andmammalian immunoglobulins.

In addition to bacterial and viral products, polysaccharide carriers arealso useful in preparing the γPGA polypeptide conjugates as disclosedherein. Polysaccharide carriers include, but are not limited to,dextran, capsular polysaccharides from microorganisms such as the Vicapsular polysaccharide from S. typhi (see, for example, U.S. Pat. No.5,204,098); Pneumococcus group 12 (12F and 12A) polysaccharides;Haemophilus influenazae type d polysaccharide; and certain plant, fruit,and synthetic oligo- or polysaccharides which are immunologicallysimilar to capsular polysaccharides, such as pectin, D-galacturonan,oligogalacturonate, or polygalacturonate (for example, as described inU.S. Pat. No. 5,738,855).

Specific, non-limiting examples of water insoluble carriers useful inpreparing the γPGA polypeptide conjugates as disclosed herein include,but are not limited to, aminoalkyl agarose (for example, aminopropyl oraminohexyl SEPHAROSE; Pharmacia Inc., Piscataway, N.J.), aminopropylglass, cross-linked dextran, and the like, to which a reactive moietycan be attached. Other carriers can be used, provided that a functionalgroup is available for covalently attaching a reactive group.

Binding of γPGA polypeptides to a carrier can be direct or via a linkerelement. Linkers can include amino acids, including amino acids capableof forming disulfide bonds, but can also include other molecules suchas, for example, polysaccharides or fragments thereof Linkers can bechosen so as to elicit their own immunogenic effect which nay be eitherthe same, or different, than that elicited by the γPGA polypeptidesand/or carriers disclosed herein. For example, such linkers can bebacterial antigens which elicit the production of antibodies to aninfectious bacteria. In such instances, for example, the linker can be aprotein or protein fragment of an infectious bacterium.

The covalent linking of the γPGA polypeptides disclosed herein to thecarrier can be carried out in any manner well known to one of skill inthe art. Conjugation methods applicable to the present disclosureinclude, by way of non-limiting example, reductive amination, diazocoupling, thioether bond, disulfide bond, amidation and thiocarbamoylchemistries. In one embodiment, the γPGA polypeptides and/or the carrierare “activated” prior to conjugation. Activation provides the necessarychemical groups for the conjugation reaction to occur. In one specific,non-limiting example, the activation step includes derivatization withadipic acid dihydrazide. In another specific, non-limiting example, theactivation step includes derivatization with the N-hydroxysuccinimideester of 3-(2-pyridyl dithio)-propionic acid (SPDP). In yet anotherspecific, non-limiting example, the activation step includesderivatization with succinimidyl 3-(bromoacetamido) propionate (SBAP).Further, non-limiting examples of derivatizing agents includesuccinimidylformylbenzoate (SFB) and succinimidyllevulinate (SLV).

Following conjugation of a γPGA polypeptide to a carrier, the γPGApolypeptide-carrier conjugate can be purified by a variety of techniqueswell known to one of skill in the art. One goal of the purification stepis to remove the unbound γPGA polypeptide from the γPGApolypeptide-carrier conjugate. One method for purification, involvingultrafiltration in the presence of ammonium sulfate, is described inU.S. Pat. No. 6,146,902. Alternatively, γPGA polypeptide-carrierconjugates can be purified away from unreacted γPGA polypeptide andcarrier by any number of standard techniques including, for example,size exclusion chromatography, density gradient centrifugation,hydrophobic interaction chromatography, or ammonium sulfatefractionation. See, for example, Anderson et al., J. Immunol.137:1181-86, 1986 and Jennings & Lugowski, J. Immunol. 127:1011-18,1981. The compositions and purity of the conjugates can be determined byGLC-MS and MALDI-TOF spectrometry.

For γPGA conjugates comprising γPGA polypeptides bound at one point to acarrier, complex structural characteristics determine optimalimmunogenicity for synthetic conjugates (see, for example, Kabat, Prog.Immunol. 5:67-85, 1983; Pozsgay et al., PNAS 96:5194-97, 1999; Lee etal., J. Immunol. 116:1711-18, 1976; and Dintzis et al., PNAS 73:3671-75,1976). γPGA polypeptide lengths must be sufficient to occupy a cognateantibody combining site. In addition, the density of the γPGApolypeptide on the carrier determines the ability of the γPGA conjugateto form both aggregates with the surface Ig receptor, and to permitinteraction of the carrier fragments with T-cells. In variousembodiments of the present disclosure, γPGA conjugates having a densityof γPGA polypeptide chains to carrier molecule of between about 5:1 toabout 32:1, such as about 8:1 to about 22:1, or about 10:1 to about15:1, are useful within the immunogenic compositions and methodsdescribed herein.

B. Analogs, Derivatives and Mimetics

In additional aspects of the disclosure, a γPGA conjugate, PA-basedimmunogen, carrier, or component of an immunogenic conjugate orcomposition of the disclosure, includes a peptide mimetic of a naturallyoccurring or synthetic agent, for example a γPGA polypeptide derivativeof B. anthracis or another Bacillus species or strain. Exemplaryconjugates and compositions are provided which comprise a peptide ornon-peptide molecule that mimics the tertiary binding structure andactivity of a selected native peptide or functional domain (for example,immunogenic region or epitope) of a γPGA polypeptide, carrier, linker,PA-based immunogen or other component of an immunogenic conjugate orcomposition of the disclosure. These peptide mimetics includerecombinantly or chemically modified peptides, as well as non-peptideagents such as small molecule drug mimetics, as further describedherein.

Certain peptidomimetic compounds are based upon the amino acid sequenceof the proteins and peptides described herein for use within thedisclosure, including sequences of bacterial toxins such as B.anthracis. PA (for example, as encoded by bases 143779 to 146073 ofGenBank Accession No. NC 007322) and LF (for example, as encoded by thecomplement of bases 149357 to 151786 of GenBank Accession No. NC007322). Typically, peptidomimetic compounds are synthetic compoundshaving a three-dimensional structure (of at least part of the mimeticcompound) that mimics, for example, the primary, secondary, and/ortertiary structural, and/or electrochemical characteristics of aselected peptide or protein, or a structural domain, active site, orbinding region (for example, a homotypic or heterotypic binding site,catalytic active site or domain, receptor or ligand binding interface ordomain) thereof. The peptide-mimetic structure or partial structure(also referred to as a peptidomimetic motif of a peptidomimeticcompound) will often share a desired biological activity with a nativepeptide or protein, as discussed herein (for example, immunogenicactivity, such as binding to an antibody or a MHC molecule to activateCD8⁺ and/or CD4⁺ T-cells). Typically, at least one subject biologicalactivity of the mimetic compound is not substantially reduced incomparison to, and is often the same as or greater than, the activity ofthe native peptide on which the mimetic was modeled.

A variety of techniques well known to one of skill in the art areavailable for constructing peptide and protein mimetics with the same,similar, increased, or reduced biological activity as the correspondingnative peptide or protein. Often these analogs, variants, derivativesand mimetics will exhibit one or more desired activities that aredistinct or improved from the corresponding native peptide or protein,for example improved characteristics of solubility, stability, and/orsusceptibility to hydrolysis or proteolysis (see, for example, Morgan &Gainor, Ann. Rep. Med. Chem. 24:243-52, 1989). In addition,peptidomimetic compounds of the disclosure can have other desiredcharacteristics that enhance their therapeutic application, such asincreased cell permeability, greater affinity and/or avidity for abinding partner, and/or prolonged biological half-life. Thepeptidomimetics of the disclosure will sometimes have a backbone that ispartially or completely non-peptide, but with side groups identical tothe side groups of the amino acid residues that occur in the peptide orprotein on which the peptidomimetic is modeled. Several types ofchemical bonds, for example, ester, thioester, thioamide, retroamide,reduced carbonyl, dimethylene and ketomethylene bonds, are known in theart to be generally useful substitutes for peptide bonds in theconstruction of protease-resistant peptidomimetics.

In one embodiment, peptides (including polypeptides) useful within thedisclosure are modified to produce peptide mimetics by replacement ofone or more naturally occurring side chains of the 20 geneticallyencoded amino acids (or D-amino acids) with other side chains, forexample with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to7-membered alkyl, amide, amide lower alkyl amide di(lower alkyl), loweralkoxy, hydroxy, carboxy and the lower ester derivatives thereof, andwith 4-, 5-, 6-, to 7-membered heterocyclics. For example, prolineanalogs can be made in which the ring size of the proline residue ischanged from a 5 membered ring to a 4, 6, or 7 membered ring. Cyclicgroups can be saturated or unsaturated, and if unsaturated, can bearomatic or non-aromatic. Heterocyclic groups can contain one or morenitrogen, oxygen, and/or sulphur heteroatoms. Examples of such groupsinclude furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl,isothiazolyl, isoxazolyl, morpholinyl (for example, morpholino),oxazolyl, piperazinyl (for example, 1-piperazinyl), piperidyl (forexample, 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl(for example, 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl,thiazolyl, thienyl, thiomorpholinyl (for example, thiomorpholino), andtriazolyl groups. These heterocyclic groups can be substituted orunsubstituted. Where a group is substituted, the substituent can bealkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.Peptides and proteins, as well as peptide and protein analogs andmimetics, can also be covalently bound to one or more of a variety ofnonproteinaceous polymers, for example, polyethylene glycol,polypropylene glycol, or polyoxyalkenes, as described in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337.

Other peptide and protein analogs and mimetics within the scope of thedisclosure include glycosylation variants, and covalent or aggregateconjugates with other chemical moieties. Covalent derivatives can beprepared by linkage of functionalities to groups which are found inamino acid side chains or at the N- or C-termini, by means which arewell known in the art. These derivatives can include, withoutlimitation, aliphatic esters or amides of the carboxyl terminus, or ofresidues containing carboxyl side chains, O-acyl derivatives of hydroxylgroup-containing residues, and N-acyl derivatives of the amino terminalamino acid or amino-group containing residues (for example, lysine orarginine). Acyl groups are selected from the group of alkyl-moietiesincluding C3 to C18 normal alkyl, thereby forming alkanoyl aroylspecies. Covalent attachment to carrier proteins, for example,immunogenic moieties, can also be employed.

In addition to these modifications, glycosylation alterations ofbiologically active peptides and proteins (including a γPGA conjugate,PA-based immunogen, carrier, or component of an immunogenic conjugate orcomposition of the disclosure) can be made, for example, by modifyingthe glycosylation patterns of a peptide during its synthesis andprocessing, or in further processing steps. In one embodiment, this isaccomplished by exposing the peptide to glycosylating enzymes derivedfrom cells that normally provide such processing, for example, mammalianglycosylation enzymes. Deglycosylation enzymes can also be successfullyemployed to yield useful modified peptides and proteins within thedisclosure. Also embraced are versions of a native primary amino acidsequence which have other minor modifications, including phosphorylatedamino acid residues, for example, phosphotyrosine, phosphoserine, orphosphothreonine, or other moieties, including ribosyl groups orcross-linking reagents.

Peptidomimetics can also have amino acid residues that have beenchemically modified by phosphorylation, sulfonation, biotinylation, orthe addition or removal of other moieties, particularly those that havemolecular shapes similar to phosphate groups. In some embodiments, themodifications will be useful labeling reagents, or serve as purificationtargets (for example, affinity ligands).

C. Specific Binding Agents

The disclosure provides specific binding agents that bind a γPGApolypeptide of B. anthracis or another Bacillus species or strain, or aγPGA conjugate as disclosed herein. The binding agent can be used topurify and detect the γPGA polypeptides, as well as for detection anddiagnosis of B. anthracis. Examples of the binding agents are apolyclonal or monoclonal antibody (including humanized monoclonalantibody), and fragments thereof, that bind to any of the γPGApolypeptides or γPGA conjugates disclosed herein.

Monoclonal or polyclonal antibodies can be raised to recognize a γPGApolypeptide and/or a γPGA conjugate as described herein, or a analog orderivative thereof. Substantially pure γPGA conjugate suitable for useas immunogen can be prepared as described above. Monoclonal orpolyclonal antibodies to the γPGA conjugate can then be prepared.

Monoclonal antibodies to the polypeptides can be prepared from murinehybridomas according to the classic method of Kohler & Milstein (Nature256:495-97, 1975), or a derivative method thereof. Briefly, a mouse isrepetitively inoculated with a few micrograms of the selected immunogen(for example, a γPGA conjugate) over a period of a few weeks. The mouseis then sacrificed, and the antibody-producing cells of the spleenisolated. The spleen cells are fused by means of polyethylene glycolwith mouse myeloma cells, and the excess unfused cells destroyed bygrowth of the system on selective media comprising aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as the enzyme-linked immunoabsorbent assay(ELISA), as originally described by Engvall (Meth. Enzymol., 70:419-39,1980), or a derivative method thereof. Selected positive clones can beexpanded and their monoclonal antibody product harvested for use.Detailed procedures for monoclonal antibody production are described inHarlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York,1999. Polyclonal antiserum containing antibodies can be prepared byimmunizing suitable animals with an immunogen comprising a γPGAconjugate.

Effective antibody production (whether monoclonal or polyclonal) isaffected by many factors related both to the antigen and the hostspecies. For example, small molecules tend to be less immunogenic thanothers and may require the use of carriers and adjuvant. Also, hostanimals vary in response to site of inoculations and dose, with eitherinadequate or excessive doses of antigen resulting in low titerantisera. Small doses (ng level) of antigen administered at multipleintradermal sites appear to be most reliable. An effective immunizationprotocol for rabbits can be found in Vaitukaitis et al. (J. Clin.Endocrinol. Metab., 33:988-91, 1971).

Booster injections can be given at regular intervals, and antiserumharvested when the antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony et al., Handbook of Experimental Immunology, Wier,D. (ed.), Chapter 19, Blackwell, 1973. A plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12μM). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher (Manualof Clinical Immunology, Ch. 42, 1980).

Antibodies of the present disclosure can be contained in blood plasma,serum, hybridoma supernatants and the like. Alternatively, theantibodies can be isolated to the extent desired by well knowntechniques in the art, such as, ion exchange chromatography, sizingchromatography, or affinity chromatography. The antibodies can bepurified so as to obtain specific classes or subclasses of antibody,such as IgM, IgG, IgA, IgG1, IgG2, IgG3, IgG4 and the like. Antibodiesof the IgG class are of use for purposes of passive protection.

Antibody fragments can be used in place of whole antibodies and can bereadily expressed in prokaryotic host cells. Methods of making and usingimmunologically effective portions of monoclonal antibodies, alsoreferred to as “antibody fragments,” are well known and include thosedescribed in Better & Horowitz, Methods Enzymol. 178:476-96, 1989;Glockshuber et al., Biochemistry 29:1362-67, 1990; and U.S. Pat. Nos.5,648,237; 4,946,778; and 5,455,030. Conditions whereby apolypeptide/binding agent complex can form, as well as assays for thedetection of the formation of a polypeptide/binding agent complex andquantitation of binding affinities of the binding agent and polypeptide,are standard in the art. Such assays can include, but are not limitedto, Western blotting, immunoprecipitation, immunofluorescence,immunocytochemistry, immunohistochemistry, fluorescence activated cellsorting, fluorescence in situ hybridization, immunomagnetic assays,ELISA, ELISPOT (Coligan et al., Current Protocols in Immunology, Wiley,N.Y., 1995), agglutination assays, flocculation assays, cell panning,and the like, as are well known to one of skill in the art.

The antibodies or antibody fragments of the present disclosure have anumber of diagnostic and therapeutic uses. For example, the antibodiesor antibody fragments can be used for passive immunotherapy, such as byadministering to a subject a therapeutically effective amount of theantibody or antibody fragments. In another example, the antibodies orantibody fragments can be used as in vitro diagnostic agents in variousimmunoassays to test for the presence of B. anthracis or anotherBacillus expressing a γPGA polypeptide in biological (for example,clinical) samples, in meat and meat products, on surfaces such as foodprocessing surfaces, or on surfaces of items subject to security testing(for example, baggage, freight, water treatment, postage handling,transportation facilities, and the like). Useful immunoassays include,but are not limited to, agglutination assays, radioimmunoassays, ELISA,fluorescence assays, Western blots and the like. In one such assay, forexample, the biological sample is contacted first with an antibody ofthe present disclosure which binds Bacillus γPGA polypeptide, and thenwith a labeled second antibody to detect the presence of a Bacillus,such as B. anthracis, to which the first antibody has bound. Such assayscan be, for example, of direct format (where a labeled first antibody isreactive with the γPGA polypeptide), an indirect format (where a labeledsecond antibody is reactive with the first antibody), a competitiveformat (such as the addition of a labeled γPGA polypeptide), or asandwich format (where both labeled and unlabelled antibody areutilized), as well as other formats well known to one of skill in theart.

Binding agents of this disclosure can be bound to a substrate (forexample, beads, tubes, slides, plates, nitrocellulose sheets, and thelike) or conjugated with a detectable moiety, or both bound andconjugated. The detectable moieties contemplated for the presentdisclosure can include, but are not limited to, an immunofluorescentmoiety (for example, fluorescein, rhodamine), a radioactive moiety (forexample, ³²P, ¹²⁵I, ³⁵S), an enzyme moiety (for example, horseradishperoxidase, alkaline phosphatase), a colloidal gold moiety, and a biotinmoiety. Such conjugation techniques are standard in the art (forexample, see Harlow and Lane, Using Antibodies: A Laboratory Manual,CSHL, New York 1999; Yang et al., Nature, 382:319-24, 1996).

D. Pharmaceutical and Immunogenic Compositions and Uses Thereof

Pharmaceutical compositions (including therapeutic and prophylacticformulations) of a γPGA conjugate and/or a PA-based immunogen are alsoencompassed by the present disclosure, and include a γPGA conjugateand/or other biologically active agent as described herein, typicallycombined together with one or more pharmaceutically acceptable vehiclesand, optionally, other therapeutic ingredients (for example,antibiotics, or anti-inflammatories).

Within the pharmaceutical compositions and methods of the disclosure,the γPGA conjugate and/or other biologically active agent can beadministered to subjects by a variety of mucosal administration modes,including by oral, rectal, intranasal, intrapulmonary, or transdermaldelivery, or by topical delivery to other surfaces. Optionally, the γPGAconjugate and/or other active agent can be administered by non-mucosalroutes, including by intramuscular, subcutaneous, intravenous,intra-atrial, intra-articular, intraperitoneal, or parenteral routes. Inother alternative embodiments, the γPGA conjugate and/or other activeagent can be administered ex vivo by direct exposure to cells, tissuesor organs originating from a subject.

To formulate pharmaceutical compositions of the present disclosure, theγPGA conjugate and/or other biologically active agent can be combinedwith various pharmaceutically acceptable additives, as well as a base orvehicle for dispersion of the γPGA conjugate and/or other biologicallyactive agent Desired additives include, but are not limited to, pHcontrol agents, such as arginine, sodium hydroxide, glycine,hydrochloric acid, citric acid, and the like. In addition, localanesthetics (for example, benzyl alcohol), isotonizing agents (forexample, sodium chloride, mannitol, sorbitol), adsorption inhibitors(for example, Tween 80), solubility enhancing agents (for example,cyclodextrins and derivatives thereof), stabilizers (for example, serumalbumin), and reducing agents (for example, glutathione) can beincluded. Adjuvants, such as aluminum hydroxide (for example, Amphogel,Wyeth Laboratories, Madison, N.J.), Freund's adjuvant, MPL™(3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton Ind.) and IL-12(Genetics Institute, Cambridge Mass.), among many other suitableadjuvants well known in the art, can be included in the compositions.When the composition is a liquid, the tonicity of the formulation, asmeasured with reference to the tonicity of 0.9% (w/v) physiologicalsaline solution taken as unity, is typically adjusted to a value atwhich no substantial, irreversible tissue damage will be induced at thesite of administration. Generally, the tonicity of the solution isadjusted to a value of about 0.3 to about 3.0, such as about 0.5 toabout 2.0, or about 0.8 to about 1.7.

The γPGA conjugate and/or other biologically active agent can bedispersed in a base or vehicle, which can include a hydrophilic compoundhaving a capacity to disperse the γPGA conjugate and/or otherbiologically active agent, and any desired additives. The base can beselected from a wide range of suitable compounds, including but notlimited to, copolymers of polycarboxylic acids or salts thereof,carboxylic anhydrides (for example, maleic anhydride) with othermonomers (for example, methyl (meth)acrylate, acrylic acid and thelike), hydrophilic vinyl polymers, such as polyvinyl acetate, polyvinylalcohol, polyvinylpyrrolidone, cellulose derivatives, such ashydroxymethylcellulose, hydroxypropylcellulose and the like, and naturalpolymers, such as chitosan, collagen, sodium alginate, gelatin,hyaluronic acid, and nontoxic metal salts thereof. Often, abiodegradable polymer is selected as a base or vehicle, for example,polylactic acid, poly(lactic acid-glycolic acid) copolymer,polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid)copolymer and mixtures thereof. Alternatively or additionally, syntheticfatty acid esters such as polyglycerin fatty acid esters, sucrose fattyacid esters and the like can be employed as vehicles. Hydrophilicpolymers and other vehicles can be used alone or in combination, andenhanced structural integrity can be imparted to the vehicle by partialcrystallization, ionic bonding, cross-linking and the like. The vehiclecan be provided in a variety of forms, including, fluid or viscoussolutions, gels, pastes, powders, mnicrospheres and films for directapplication to a mucosal surface.

The γPGA conjugate and/or other biologically active agent can becombined with the base or vehicle according to a variety of methods, andrelease of the γPGA conjugate and/or other biologically active agent canbe by diffusion, disintegration of the vehicle, or associated formationof water channels. In some circumstances, the γPGA conjugate and/orother biologically active agent is dispersed in microcapsules(microspheres) or nanocapsules (nanospheres) prepared from a suitablepolymer, for example, isobutyl 2-cyanoacrylate (see, for example,Michael et al., J. Pharmacy Pharmacol. 43:1-5, 1991), and dispersed in abiocompatible dispersing medium, which yields sustained delivery andbiological activity over a protracted time.

The compositions of the disclosure can alternatively contain aspharmaceutically acceptable vehicles substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, and triethanolamineoleate. For solid compositions, conventional nontoxic pharmaceuticallyacceptable vehicles can be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like.

Pharmaceutical compositions for administering the γPGA conjugate and/orother biologically active agent can also be formulated as a solution,microemulsion, or other ordered structure suitable for highconcentration of active ingredients. The vehicle can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycol, and thelike), and suitable mixtures thereof. Proper fluidity for solutions canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of a desired particle size in the case of dispersibleformulations, and by the use of surfactants. In many cases, it will bedesirable to include isotonic agents, for example, sugars, polyalcohols,such as mannitol and sorbitol, or sodium chloride in the composition.Prolonged absorption of the γPGA conjugate and/or other biologicallyactive agent can be brought about by including in the composition anagent which delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, the γPGA conjugate and/or other biologicallyactive agent can be administered in a time release formulation, forexample in a composition which includes a slow release polymer. Thesecompositions can be prepared with vehicles that will protect againstrapid release, for example a controlled release vehicle such as apolymer, microencapsulated delivery system or bioadhesive gel. Prolongeddelivery in various compositions of the disclosure can be brought aboutby including in the composition agents that delay absorption, forexample, aluminum monostearate hydrogels and gelatin. When controlledrelease formulations are desired, controlled release binders suitablefor use in accordance with the disclosure include any biocompatiblecontrolled release material which is inert to the active agent and whichis capable of incorporating the γPGA conjugate and/or other biologicallyactive agent. Numerous such materials are known in the art. Usefulcontrolled-release binders are materials that are metabolized slowlyunder physiological conditions following their delivery (for example, ata mucosal surface, or in the presence of bodily fluids). Appropriatebinders include, but are not limited to, biocompatible polymers andcopolymers well known in the art for use in sustained releaseformulations. Such biocompatible compounds are non-toxic and inert tosurrounding tissues, and do not trigger significant adverse sideeffects, such as nasal irritation, immune response, inflammation, or thelike. They are metabolized into metabolic products that are alsobiocompatible and easily eliminated from the body.

Exemplary polymeric materials for use in the present disclosure include,but are not limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolyzable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids and polylactic acids, poly(DL-lacticacid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), andpoly(L-lactic acid-co-glycolic acid). Other useful biodegradable orbioerodable polymers include, but are not limited to, such polymers aspoly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid),poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyricacid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) (for example, L-leucine,glutamic acid, L-aspartic acid and the like), poly(ester urea),poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,polyorthoesters, polycarbonate, polymaleamides, polysaccharides, andcopolymers thereof. Many methods for preparing such formulations arewell known to those skilled in the art (see, for example, Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978). Other useful formulations includecontrolled-release microcapsules (U.S. Pat. Nos. 4,652,441 and4,917,893), lactic acid-glycolic acid copolymers useful in makingmicrocapsules and other formulations (U.S. Pat. Nos. 4,677,191 and4,728,721) and sustained-release compositions for water-soluble peptides(U.S. Pat No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterileand stable under conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the γPGA conjugate and/orother biologically active agent in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated herein, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the γPGA conjugate and/or other biologicallyactive agent into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated herein.In the case of sterile powders, methods of preparation include vacuumdrying and freeze-drying which yields a powder of the γPGA conjugateand/or other biologically active agent plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theprevention of the action of microorganisms can be accomplished byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

In accordance with the various treatment methods of the disclosure, theγPGA conjugate and/or other biologically active agent can be deliveredto a subject in a manner consistent with conventional methodologiesassociated with management of the disorder for which treatment orprevention is sought. In accordance with the disclosure herein, aprophylactically or therapeutically effective amount of the γPGAconjugate and/or other biologically active agent is administered to asubject in need of such treatment for a time and under conditionssufficient to prevent, inhibit, and/or ameliorate a selected disease(for example, anthrax) or condition or one or more symptom(s) thereof.

Typical subjects intended for treatment with the compositions andmethods of the present disclosure include humans, as well as non-humanprimates and other animals. To identify subjects for prophylaxis ortreatment according to the methods of the disclosure, accepted screeningmethods are employed to determine risk factors associated with atargeted or suspected disease of condition (for example, anthrax) asdiscussed herein, or to determine the status of an existing disease orcondition in a subject. These screening methods include, for example,conventional work-ups to determine environmental, familial,occupational, and other such risk factors that may be associated withthe targeted or suspected disease or condition, as well as diagnosticmethods, such as various ELISA and other immunoassay methods, which areavailable and well known in the art to detect and/or characterizedisease-associated markers. These and other routine methods allow theclinician to select patients in need of therapy using the methods andpharmaceutical compositions of the disclosure. In accordance with thesemethods and principles, a γPGA conjugate and/or other biologicallyactive agent can be administered according to the teachings herein as anindependent prophylaxis or treatment program, or as a follow-up, adjunctor coordinate treatment regimen to other treatments, including surgery,vaccination, immunotherapy, hormone treatment, cell, tissue, or organtransplants, and the like.

The γPGA conjugates can be used in coordinate vaccination protocols orcombinatorial formulations with PA-based immunogens to enhance an immuneresponse elicited by a PA-based immunogen alone. In exemplaryembodiments, γPGA-rPA induces both anti-PA and anti-γPGA immuneresponses. In other embodiments, novel combinatorial immunogeniccompositions and coordinate immunization protocols employ separateimmunogens or formulations, each directed toward eliciting an anti-PA oran anti-γPGA immune response. Separate immunogens that elicit theanti-PA or anti-γPGA immune response can be combined in a polyvalentimmunogenic composition administered to a subject in a singleimmunization step, or they can be administered separately (in monovalentimmunogenic compositions) in a coordinate immunization protocol.Typically, when the anti-PA and anti-γPGA immunogens are administeredseparately, they are administered coordinately, in close temporalsequence (for example, the anti-PA immunogen will be administered hours,one or two days, or within a week or two, prior to administration of theanti-γPGA immunogen, or vice versa).

The administration of the γPGA conjugate and/or other biologicallyactive agent of the disclosure can be for either prophylactic ortherapeutic purpose. When provided prophylactically, the γPGA conjugateand/or other biologically active agent is provided in advance of anysymptom. The prophylactic administration of the γPGA conjugate and/orother biologically active agent serves to prevent or ameliorate anysubsequent infection. When provided therapeutically, the γPGA conjugateand/or other biologically active agent is provided at (or shortly after)the onset of a symptom of disease or infection. The γPGA conjugateand/or other biologically active agent of the disclosure can thus beprovided prior to the anticipated exposure to B. anthracis or anotherBacillus, so as to attenuate the anticipated severity, duration orextent of an infection and/or associated disease symptoms, afterexposure or suspected exposure to the bacteria, or after the actualinitiation of an infection.

For prophylactic and therapeutic purposes, the γPGA conjugate and/orother biologically active agent disclosed herein can be administered tothe subject in a single bolus delivery, via continuous delivery (forexample, continuous transdermal, mucosal or intravenous delivery) overan extended time period, or in a repeated administration protocol (forexample, by an hourly, daily or weekly, repeated administrationprotocol). The therapeutically effective dosage of the γPGA conjugateand/or other biologically active agent can be provided as repeated doseswithin a prolonged prophylaxis or treatment regimen, that will yieldclinically significant results to alleviate one or more symptoms ordetectable conditions associated with a targeted disease or condition asset forth herein. Determination of effective dosages in this context istypically based on animal model studies followed up by human clinicaltrials and is guided by administration protocols that significantlyreduce the occurrence or severity of targeted disease symptoms orconditions in the subject. Suitable models in this regard include, forexample, murine, rat, porcine, feline, non-human primate, and otheraccepted animal model subjects known in the art. Alternatively,effective dosages can be determined using ice vitro models (for example,immunologic and histopathologic assays). Using such models, onlyordinary calculations and adjustments are required to determine anappropriate concentration and dose to administer a therapeuticallyeffective amount of the γPGA conjugate and/or other biologically activeagent (for example, amounts that are effective to elicit a desiredimmune response or alleviate one or more symptoms of a targeteddisease). In alternative embodiments, an effective amount or effectivedose of the γPGA conjugate and/or biologically active agent may simplyinhibit or enhance one or more selected biological activities correlatedwith a disease or condition, as set forth herein, for either therapeuticor diagnostic purposes.

The actual dosage of the γPGA conjugate and/or other biologically activeagent will vary according to factors such as the disease indication andparticular status of the subject (for example, the subject's age, size,fitness, extent of symptoms, susceptibility factors, and the like), timeand route of administration, other drugs or treatments beingadministered concurrently, as well as the specific pharmacology of theγPGA conjugate and/or other biologically active agent for eliciting thedesired activity or biological response in the subject. Dosage regimenscan be adjusted to provide an optimum prophylactic or therapeuticresponse. A therapeutically effective amount is also one in which anytoxic or detrimental side effects of the γPGA conjugate and/or otherbiologically active agent is outweighed in clinical terms bytherapeutically beneficial effects. A non-limiting range for atherapeutically effective amount of a γPGA conjugate and/or otherbiologically active agent within the methods and formulations of thedisclosure is about 0.01 mg/kg body weight to about 10 mg/kg bodyweight, such as about 0.05 mg/kg to about 5 mg/kg body weight, or about0.2 mg/kg to about 2 mg/kg body weight. The antibodies of the presentdisclosure will typically be administered in a dosage ranging from about1 mg/kg body weight to about 10 mg/kg body weight of the subject,although a lower or higher dose can be administered.

Upon administration of a γPGA conjugate (for example, γPGA-PA) orrelated immunogenic composition of the disclosure (for example, viainjection, aerosol, oral, topical or other route), the immune system ofthe subject typically responds to the immunogenic composition byproducing antibodies specific for γPGA and/or PA. Such a responsesignifies that an immunologically effective dose of the γPGA conjugateor related immunogenic composition was delivered. An immunologicallyeffective dosage can be achieved by single or multiple administrations(including, for example, multiple administrations per day), daily, orweekly administrations. For each particular subject, specific dosageregimens can be evaluated and adjusted over time according to theindividual need and professional judgment of the person administering orsupervising the administration of the γPGA conjugate and/or otherbiologically active agent. In some embodiments, the antibody response ofa subject administered the compositions of the disclosure will bedetermined in the context of evaluating effective dosages/immunizationprotocols. In most instances it will be sufficient to assess theantibody titer in serum or plasma obtained from the subject. Decisionsas to whether to administer booster inoculations and/or to change theamount of the composition administered to the individual can be at leastpartially based on the antibody titer level. The antibody titer levelcan be based on, for example, an immunobinding assay which measures theconcentration of antibodies in the serum which bind to a specificantigen, for example, γPGA and/or PA. The ability to neutralize in vitroand in vivo biological effects of the B. anthracis can also be assessedto determine the effectiveness of the treatment.

Dosage can be varied by the attending clinician to maintain a desiredconcentration at a target site (for example, the lungs or systemiccirculation). Higher or lower concentrations can be selected based onthe mode of delivery, for example, trans-epidermal, rectal, oral,pulmonary, or intranasal delivery versus intravenous or subcutaneousdelivery. Dosage can also be adjusted based on the release rate of theadministered formulation, for example, of an intrapulmonary spray versuspowder, sustained release oral versus injected particulate ortransdermal delivery formulations, and so forth. To achieve the sameserum concentration level, for example, slow-release particles with arelease rate of 5 nanomolar (under standard conditions) would beadministered at about twice the dosage of particles with a release rateof 10 nanomolar.

The methods of using γPGA conjugates, and the related compositions andmethods of the disclosure, are useful in increasing resistance to,preventing, ameliorating, and/or treating infection and disease causedby bacilli in animal hosts, and other, in vitro applications. Inexemplary embodiments, the methods and compositions are useful inincreasing resistance to, preventing, ameliorating, and/or treatinginfection and disease caused by B. anthracis infection in animals andhumans. These immunogenic compositions can be used for activeimmunization for prevention of B. anthracis infection, and forpreparation of immune antibodies. In one embodiment, the immunogeniccompositions and methods are designed to confer specific immunityagainst infection with B. anthracis, and to induce antibodies specificto B. anthracis γPGA. The immunogenic compositions are composed ofnon-toxic components, suitable for infants, children of all ages, andadults.

The methods of the disclosure are broadly effective for treatment andprevention of bacterial disease and associated inflammatory, autoimmune,toxic (including shock), and chronic and/or lethal sequelae associatedwith bacterial infection. In selected embodiments, one or more symptomsor associated effects of exposure to and/or infection with anthraxis/are prevented or treated by administration to a mammalian subject atrisk of acquiring anthrax, or presenting with one or more anthraxsymptom(s), of an effective amount of a γPGA conjugate of thedisclosure. Therapeutic compositions and methods of the disclosure forprevention or treatment of toxic or lethal effects of bacterialinfection are applicable to a wide spectrum of infectious agents.Non-lethal toxicities that will be ameliorated by these methods andcompositions can include fatigue syndromes, inflammatory/autoimmunesyndromes, hypoadrenal syndromes, weakness, cognitive symptoms andmemory loss, mood symptoms, neurological and pain syndromes andendocrine symptoms. Any significant reduction or preventive effect ofthe γPGA conjugate with respect to the foregoing disease condition(s) orsymptom(s) administered constitutes a desirable, effective property ofthe subject composition/method of the disclosure.

The compositions and methods of the disclosure are particularly usefulfor treatment and prevention of infection and toxic/morbidity effects ofexposure to anthrax and/or other disease- or illness-causing bacilli.Additional embodiments of the disclosure are directed to diagnosticcompositions and methods to identify individuals at risk for exposure,infection, toxic effects, or long term deleterious effects of exposureto pathogenic bacteria, for example B. anthracis. In additional aspectsof the disclosure, the methods and compositions disclosed herein areuseful for identification of environmental agents, including B.anthracis and other bacilli expressing a γPGA, including food-bornepathogenic bacilli. Certain individuals exposed to small amounts ofbacterial products, such as those derived from B. anthracis, presentingcertain genetic or physiological backgrounds, are predisposed todevelopment of chronic syndromes, including fatigue syndromes,inflammatory/autoimmune syndromes, hypoadrenal syndromes, weakness,cognitive symptoms and memory loss, mood symptoms, neurological and painsyndromes and endocrine symptoms. In this context, the methods andcompositions of the disclosure are employed to detect, and alternativelyto treat and/or ameliorate, such ubiquitous environmental exposures andassociated symptoms. For example, antibodies of the disclosure providefor screening for γPGA in mammalian subjects or food products at risk ofcontact/infection with a Bacillus that expresses a γPGA.

In related embodiments, the disclosure provides compositions, includingbut not limited to, mammalian serum, plasma, and immunoglobulinfractions, which contain antibodies that are immunoreactive with a γPGAof B. anthracis or another Bacillus species or strain. These antibodiesand antibody compositions can be useful to prevent, treat, and/orameliorate infection and disease caused by the microorganism. Thedisclosure also provides such antibodies in isolated form. In exemplaryembodiments, high titer anti-γPGA sera, antibodies isolated therefrom,or monoclonal antibodies, can be used for therapeutic treatment forpatients with infection by B. anthracis or another Bacillus species orstrain. Antibodies elicited by the agents of this disclosure can be usedfor the treatment of established B. anthracis or other Bacillusinfections, and can also be useful in providing passive protection to anindividual exposed to B. anthracis or another Bacillus.

The instant disclosure also includes kits, packages and multi-containerunits containing the herein described pharmaceutical compositions,active ingredients, and/or means for administering the same for use inthe prevention and treatment of anthrax and other bacterial diseases andother conditions in mammalian subjects. Kits for diagnostic use are alsoprovided. In one embodiment, these kits include a container orformulation that contains one or more of the γPGA conjugates and/orother active agent described herein. In one example, this component isformulated in a pharmaceutical preparation for delivery to a subject.The γPGA conjugate and/or other biologically active agent is/areoptionally contained in a bulk dispensing container or unit ormulti-unit dosage form. Optional dispensing means can be provided, forexample a pulmonary or intranasal spray applicator. Packaging materialsoptionally include a label or instruction indicating for what treatmentpurposes (for example, anthrax) and/or in what manner the pharmaceuticalagent packaged therewith can be used.

The subject matter of the present disclosure is further illustrated bythe following non-limiting Examples.

EXAMPLES Example 1 Materials and Methods

Bacterial Strains

B. pumilus, strain Sh18 (Goodman et al., Biochem. 7:706-10, 1968), andB. anthracis strain A34, a pX01⁻, pX02⁺ variant derived from the Amesstrain by repeated passage at 43° C., are described by Klein et al.(Science 138:1331-33, 1962).

Poly-γ-glutamic Acid

γPGA was extracted from culture supernatants of B. anthracis or B.pumilus by acidification to pH 1.5, precipitation with ethanol, andpassage through a 2×100 cm Sepharose CL-4B column in 0.2 M NaCl(Myerowitz et al., Infect. Immun. 8:896-900, 1973). The composition ofeach γPGA was confirmed by ¹H-NMR and ¹³C-NMR and their enantiomericcompositions were determined by GLC-MS spectroscopy.

Analyses

Amino acid analyses were conducted by GLC-MS after hydrolysis with 6 NHCl, 150° C., 1 hour, derivatization to heptafluorobutyryl R-(−)isobutylesters and assayed with a Hewlett-Packard apparatus (Model HP 6890) witha HP-5 0.32×30 mm glass capillary column, temperature programming at 8°C./min, from 125° C. to 250° C. in the electron ionization (106 eV) mode(MacKenzie, J. Assoc. Off. Anal. Chem. 70:151-60, 1987). Under theseconditions, D-glutamic acid is separated from the L-enantiomer so thatthe ratio of each can be calculated based on the ratio of D-glutamicacid relative to L-glutamic acid residues in the protein (FIG. 1). Thenumber of peptide chains in L-peptide conjugates was calculated by therelative increase of total L-glutamic acid relative to aspartic acid.Protein concentration was measured by the method of Lowry et al. (J.Biol. Chem. 193:266-73, 1951), free ε amino groups by Fields' assay(Biochem. J. 124:581-90, 1971), thiolation by release of 2-pyridylthiogroups (A₃₄₃) (Carlsson et al, Biochem. J. 173:723-37, 1978), andhydrazide as reported by Schneerson et al. (J. Exp. Med. 152:361-76,1980). SDS-PAGE employed 14% gels according to the manufacturer'sinstructions. Double immunodiffusion was performed in 1.0% agarose gelin PBS.

MALDI-TOF

Mass spectra were obtained with a PerSeptive BioSystems Voyager EliteDE-STR MALDI-TOF instrument (PE Biosystems, Framingham, Mass.) operatedin the linear mode, 25 kV accelerating voltage and a 300 nanosecond ionextraction delay time. Samples for analysis were prepared by a“sandwich” of matrix and analyte. First, 1 μl matrix (saturated solutionof sinnapinic acid made in 1:1 CH₃CN and 0.1% trifluroacetic acid) wasdried on the sample stage. Second, 1 μl of sample and an additional 1 μlof matrix was applied. After the “sandwich” was dried, the sample wasplaced in the mass spectrometer.

Antigens

BSA (Sigma Chemical Co., St. Louis, Mo.) was dialyzed againstpyrogen-free water, sterile-filtered, and freeze-dried. RecombinantProtective Antigen from B. anthracis and recombinant exotoxin A from P.aeruginosa were prepared and characterized as described by Ramirez etal. (J. Ind. Microbiol. Biotechnol. 28:232-38, 2002) and Johansson etal. (J. Biotechnol. 48:9-14, 1996). Exemplary synthetic polypeptides ofγPGA (AnaSpec, San Jose, Calif.) were synthesized by the method ofMerrifield, with lengths of 5, 10, 15, or 20 residues. Their purity andauthenticity were verified by GLC-MS, LC-MS and MALDI-TOF. γPGApolypeptides were bound to carrier proteins at either the C- or theN-termini (—C indicates that the C-terminus is free; N— indicates thatthe amino-terminus is free). All reactions were conducted in a pH statunder argon. Type I: NBrAc-Gly₃-γDPGA_(n)-COOH(Br-Gly₃-γDPGA_(n)-C)NBrAc-Gly₃₋γLPGA_(n)-COOH(Br-Gly₃-γLPGA_(n)-C) Type II:NAc-L-Cys-Gly₃-γDPGA_(n)-COOH(Cys-Gly₃-γDPGA_(n)-C)NAc-L-Cys-Gly₃-γLPGA_(n)-COOH(Cys-Gly₃-γLPGA_(n)-C) Type III:NAc-γDPGA_(n)-Gly₃-L-Cys-CONH₂(N-γDPGA_(n)-Gly₃-Cys)NAc-γLPGA_(n)-Gly₃-L-Cys-CONH₂(N-γLPGA_(n)-Gly₃-Cys) Type IV:CHO-Gly₃-γDPGA_(n)-COOH Type V: NAc-γDPGA_(n)-Gly₃-CO-AHNAc-γDPGA_(n)-CO-AH Type VI: NAc-γDPGA_(n)-Cys-CONH₂Conjugation of BSA, rEPA and rPA with B. anthracis γDPGA and B. pumilusγDLPGA

BSA, rEPA and rPA were derivatized with adipic acid dihydrazide withmodifications (Schneerson et al., J. Exp. Med. 152:361-76, 1980). The pHwas maintained at 7.0 and 0.1 M EDAC used. The products, BSA-AH, rEPA-AHand rPA-AH, contained 2.0-4.8% hydrazide.

γPGA was bound to rPA-AH or rEPA-AH with 0.01 M EDAC, the reactionmixture passed through a 1×90 cm Sephacryl S-1000 column in 0.2 M NaCl,and fractions reacting with anti-PA and anti-γDPGA by an identity linewere pooled.

Conjugation of Type I Peptide with rPA via Thioether Bond

Step 1: Derivatization of BSA, rEPA and rPA with SPDP

To rPA (30 mg) in 1.5 ml of Buffer A′ (PBS, 3% glycerol, 0.005 M EDTA,pH 7.6), SPDP (10 mg) in 50 μl dimethyl sulfoxide (DMSO) was added in 10μl aliquots and reacted for 1 hour at pH 7.6. The product,2-pyridyldithio-propionyl-rPA (PDP-rPA) was passed through a 1×48 cmSephadex G-50 column in Buffer A (PBS, 0.05% glycerol 0.005 M EDTA, pH7.6), and protein-containing fractions were pooled and assayed forthiolation, antigenicity, and molecular mass (Carlsson et al, Biochem.J. 173:723-37, 1978).

Step 2: Conjugation of PDP-Protein with Type I Peptide

PDP-protein (24 mg) in 2 ml Buffer A was treated with 50 mMdithiothreitol for 30 minutes at room temperature and passed through a1×48 cm Sephadex G-50 column in Buffer A. Fractions containing the3-thiopropionyl-ε-Lys-NH₂-rPA (rPA-SH) were collected, concentrated to1.5 ml and glycerol added to a final concentration of 3%.Br-Gly₃-γ-DPGA_(n)-C, 10 mg in 1 ml of Buffer A, was adjusted to pH 7.6and rPA-SH added, incubated for 1 hour at room temperature (Inman etal., Bioconj. Chem. 2:458-63, 1991), transferred to a vial, capped andtumbled overnight at room temperature. Bromoacetamide, 0.5 mg in 50 μlBuffer A, was added to block unreacted thiols. After 30 minutes, thereaction mixture was passed through a 1×90 cm Sephacryl S-200 column inBuffer B (0.01 M phosphate, 0.2 M NaCl, 0.05% glycerol, pH 7.2).Fractions containing protein-γPGA were pooled and assayed for peptideand protein concentration, antigenicity, and molecular mass.

Products:

BSA contained 60, rPA contained 58 and rEPA contained 15 moles Lys permole of protein, respectively. Under these conditions, 28 of 60ε-Lys-NH₂ of BSA, 50-55 of 58 of rPA and 15 of 15 of rEPA werederivatized with SPDP with retention of their antigenicity. Conjugationof BSA-SH, rPA-SH and rEPA-SH with Type I peptides yielded:

-   -   BSA-SH/Gly₃-γDPGA_(n)-C    -   BSA-SH/Gly₃-γLPGA_(n)-C    -   rEPA-SH/Gly₃-γDPGA_(n)-C    -   rPA-SH/Gly₃-γDPGA_(n)-C        Conjugation with Type II, III and VI Peptides

Step 1: Derivatization of Protein with SBAP

rPA or rEPA (30 mg) in 1.5 ml of Buffer A′ was adjusted to pH 7.2. SBAP(11 mg) in 50 μl DMSO was added in 10 μl aliquots (Inman et al.,Bioconj. Chem. 2:458-63, 1991). After 60 minutes, the reaction mixturewas passed through a 1×90 cm Sepharose CL-6B column in Buffer B.Fractions containing bromoacetamidopropionyl-ε-Lys-NH-rPA (Br-rPA) werecollected and assayed for protein, free —NH₂, antigenicity, andmolecular mass.

Step 2: Conjugation of Br-Protein with Type II, III and VI Peptides

Type II, III or VI peptides, 5 to 15 mg in Buffer A, were adjusted to pH7.6 with 1 N NaOH. Br-protein (25 mg) in 1.5 ml Buffer A′ was added.After 1 hour, the reaction mixture was transferred to a vial, capped,and tumbled overnight at room temperature. γ-mercaptoethanol (1 μl) wasadded to quench the remaining bromoacetyl groups in Br-protein. After 30minutes, the reaction mixture was passed through 1×90 cm Sepharose CL-6Bcolumn in Buffer B. Fractions containing protein-γPGA were pooled andassayed for peptide and protein concentration, antigenicity, andmolecular mass.

Products:

Under these conditions, 50-55 of 58 and 15 of 15 residues of ε-Lys-NH₂of rPA and rEPA, respectively, were modified with SBAP. rPA_(form) had30 out of 58 ε-Lys-NH₂ free, and derivatization with SBAP convertedessentially all 30 γ-Lys-NH₂ into the bromoacylated derivative,Br-rPA_(form).

Conjugation of Br-rPA and Br-rEPA with Type II peptides yielded 4conjugates:

-   -   rPA/S-Cys-Gly₃-γDPGA_(n)-C    -   rPA/S-Cys-Gly₃-γLPGA_(n)-C    -   rEPA/S-Cys-Gly₃-γDPGA_(n)-C    -   rEPA/S-Cys-Gly₃-γLPGA_(n)-C

Conjugation of Br-rPA and Br-rEPA with Type III peptides yielded 4conjugates:

-   -   N-γDPGA_(n)-Gly₃-Cys-S/rPA    -   N-γLPGA_(n)-Gly₃-Cys-S/rPA    -   N-γDPGA_(n)-Gly₃-Cys-S/rEPA    -   N-γLPGA_(n)-Gly₃-Cys-S/rEPA

All eight conjugates precipitated with an identity reaction with theirprotein and γPGA, antisera by immunodiffusion. Representative analysisby MALDI-TOF is shown in FIG. 2.

Conjugation of Br-rEPA with Type VI peptide yielded:

-   -   rEPA/Cys-γDPGA_(n)-N

Conjugation of Br-rPA_(form) with the N-γDPGA_(n)-Gly₃-Cys Type IIIpeptide yielded:

-   -   rPA_(form)/Cys-Gly₃-γDPGA_(n)-N        Conjugation of Type IV Peptide with BSA, rEPA and rPA via        Hydrazone Linkage

4-formylbenzoyl-γDPGA (CHO-γDPGA) was bound to BSA-AH, rEPA-AH or rPA-AHin phosphate buffer, pH 7.0, at a molar ratio of CHO-γDPGA to carrierprotein-AH of 2:1 for 24-48 hours at room temperature. The reactionmixture was passed through a 1×90 cm Sepharose CL-6B column in 0.2 Mphosphate buffer, pH 7.0, and fractions reacting with anti-carrierprotein and anti-γDPGA antibodies were pooled.

Conjugation of BSA-AH, rEPA-AH or rPA-AH with Type IV peptides yielded:

-   -   BSA-AH/CHO-Gly₃-γDPGA_(n)-C    -   rEPA-AH/CHO-Gly₃-γDPGA_(n)-C    -   rPA-AH/CHO-Gly₃-γDPGA_(n)-C        Conjugation of Type V Peptide with BSA, rEPA, rPA, rPA_(form)        via Hydrazone Linkage

Step 1: Derivatization of BSA, rEPA, rPA, or rPA_(form) with SFB

To BSA (30 mg) in 1.2 ml of Buffer A containing 1% glycerol, SFB (7.5mg) in 100 μl DMSO was added and reacted for 1 hour at pH 7.6. Theproduct, 4-formylbenzoyl-BSA (CHO-BSA), was passed through a 1×48 cmSephadex G-50 column in Buffer A. Protein containing fractions werepooled and assayed for the presence of benzoylaldehyde, antigenicity andprotein concentration. For rPA, rEPA and rPA_(form), derivatization withSFB was performed using 4 mg/ml rPA, rEPA and rPA_(form), respectively.

Step 2: Conjugation of CHO-BSA, CHO-rEPA, CHO-rPA or CHO-rPA_(form) withType V Peptides

To CHO-BSA, CHO-rEPA, CHO-rPA or CHO-rPA_(form) (20 mg) in 1.25 ml ofBuffer A, 20 mg of Type V peptides dissolved in 400 μl of 1M phosphatebuffer, pH 7.4, was added. The pH of the reaction mixture was adjustedto 7.0 and incubated for 48-72 hours at room temperature. The mixturewas passed through a 1×90 cm Sepharose CL-6B column in Buffer A, andfractions reacting with anti-carrier protein and anti-γDPGA antibodieswere pooled.

Products:

rPA_(form) had 30 out of 58 ε-Lys-NH₂ free (28 Lys were modified by theformaldehyde treatment), and the derivatization with SFB convertedessentially all 30 ε-Lys-NH₂ into 4-formylbenzoyl-rPA_(form)(CHO-rPA_(form)). Conjugation of CHO-BSA, CHO-rEPA, CHO-rPA orCHO-rPA_(form) with Type V peptides yielded:

-   -   BSA-CHO/AH-Gly₃-γDPGA_(n)-N    -   rEPA-CHO/AH-γDPGA_(n)-N    -   rPA-CHO/AH-γDPGA_(n)-N    -   rPA_(form)-CHO/AH-Gly₃-γDPGA_(n)-N        Conjugation of BSA-CHO/AH with Type IV Peptide via Hydrazone        Linkage

Step 1: Derivatization of BSA with SLV

To BSA (56 mg) in 2.0 ml of Buffer A was added SLV (20 mg) in 200 μlDMSO at pH 7.6 and reacted for 1 hour at room temperature. The product,BSA-LV-CHO, was passed through a 1×48 cm Sephadex G-50 column in BufferA. Protein containing fractions were pooled and assayed for proteinconcentration.

Step 2: Derivatization of BSA-LV-CHO with ADH

BSA-LV-CHO (35 mg) in 1.5 ml of 0.2 M phosphate buffer, pH 6.0, wasreacted with adipic acid dihydrazide (250 mg) at pH 6.0 in the presenceof 100 μl of borane-hydride-pyridine complex (800 μmoles) for 48 hours.The product, BSA-LV-CHO/AH, was passed through a 1×48 cm Sephadex G-50column in Buffer A. BSA containing fractions were collected, analyzedfor protein concentration, and the degree of -AH derivatization.

Step 3: Conjugation of BSA-LV-CHO/AH with Type IV Peptide

BSA-LV-CHO/AH (20 mg) in 1.5 ml of 0.2 M phosphate buffer, pH 6.0, wasmixed with 10 mg Type IV peptide, pH 6.0. After 60 minutes, 100 μl ofborane-hydride-pyridine complex (800 μmoles) was added, and after 48hours the product was passed through a 1×48 cm Sephadex G-50 column inBuffer A. Fractions reacting with anti-BSA and anti-γDPGA antibodieswere pooled.

Conjugation of BSA-LV-CHO/AH with Type IV peptide yielded:

-   -   BSA-SL-AH/CHO-Gly₃-γDPGA_(n)-C        Immunization

Five- to six-week old female NIH GP mice were immunized s.c. 3 times at2-week intervals with 2.5 μg γPGA as a conjugate in 0.1 ml of PBS, andgroups of 10 mice were exsanguinated 7 days after the second or thirdinjections (Schneerson et al, J. Exp. Med. 152:361-76, 1980). Controlsreceived PBS.

Antibodies

Serum IgG antibodies were measured by ELISA (Taylor et al, Infect.Immun. 61:3678-87, 1993). Nunc Maxisorb plates were coated with γDPGA,20 μg/ml PBS or 4 μg rPA/ml PBS. Plates were blocked with 0.5% BSA (orwith 0.5% HSA for assay of BSA conjugates) in PBS for 2 hours at roomtemperature. A MRX Dynatech reader was used. Antibody levels werecalculated relative to standard sera: for γDPGA, a hyperimmune murineserum, prepared by multiple i.p. injections of formalin-treated B.anthracis strain A34 and assigned a value of 100 ELISA units (EU), forPA a mAb containing 4.7 mg Ab/ml (Little et al., Infect. Immun.56:1807-13, 1988). Results were computed with an ELISA data processingprogram provided by the Biostatistics and Information Management Branch,CDC (Plikaytis et al., User's Manual 12 CDC, Version 1.00, 1996). IgGlevels are expressed as geometric mean (GM).

Opsonophagocytosis

Spores of B. anthracis, strain A34, were maintained at 5×10⁸ spores perml in 1% phenol. The human cell line, HL-60 (CCL240, ATCC, Rockville,Md.) was expanded and differentiated by dimethyl formamide into 44%myelocytes and metamyelocytes, and 53% band and polymorphonuclearleukocytes (PMLs). PMLs were at an effector/target cell ratio of 400:1.PMLs were centrifuged and resuspended in opsonophagocytosis buffer(Hanks' buffer with Ca²⁺, Mg²⁺ and 0.1% gelatin (Life Technologies,Grand Island, N.Y.)) at 2×10⁷ cells per ml. Spores were cultured at5×10⁷ spores per ml for 3 hours in 20% CO₂, and diluted to 5×10⁴ sporesper ml. Sera were diluted 2-fold with 0.05 ml of opsonophagocytosisbuffer, and 0.02 ml (containing approximately 10³ bacteria) were addedto each well of a 24-well tissue culture plate (Falcon, Franklin Lakes,N.J.). The plates were incubated at 37° C. in 5% CO₂ for 15 min. A 0.01ml of aliquot of colostrum-deprived baby calf serum (complement) and0.02 ml of HL-60 suspension containing 4×10⁵ cells was added to eachwell, and incubated at 37° C. in 5% CO₂ with mixing at 220 rpm for 45minutes. A 0.01 ml aliquot from each well was added to tryptic soy agarat 50° C., and CFU determined the next morning.

Opsonophagocytosis was defined by ≧50% killing compared with the growthin control wells (Romero-Steiner et al., Clin. Diagn. Lab. Immunol.4:415-33, 1997).

Statistics

ELISA values are expressed as the GM. An unpaired t test was used tocompare GMs in different groups of mice.

Example 2 Serum IgG Anti-γDPGA Antibodies

This example demonstrates that conjugates of B. anthracis γDPGA and ofB. pumilus γD/LPGA elicited IgG anti-γDPGA antibodies.

Native γDPGA from the capsule of B. anthracis elicited trace levels ofantibodies after the third injection (Table 1). All the conjugates, incontrast, elicited IgG anti-γDPGA antibodies after two injections (Table1). Conjugates of B. anthracis γDPGA and of B. pumilus γD(60%)/L(40%)PGAelicited IgG anti-γDPGA antibodies of intermediate levels after twoinjections with a booster after the third (Table 1). However,precipitates were formed during the synthesis of both conjugates,resulting in low yields. This problem was not encountered when preparingthe synthetic γPGA conjugates.

The highest levels of anti-γDPGA antibodies were achieved with peptidedecamers at a density (peptide chains to carrier molecule) of 16:1 forrPA/Cys-Gly₃-γDPGA₁₀-C, and of 11:1 and 14:1 for rPA-SH/Gly₃-γDPGA₁₀-C(Table 1). rPA was a more effective carrier than rEPA or BSA (Table 1).With the exception of rPA-SH/Gly₃-γDPGA₁₀-C, with 11 chains per carrierprotein, all conjugates elicited a rise in anti-γDPGA antibodies afterthe third injection (Table 1). Conjugates prepared with L peptides boundat either the C- or N-terminus induced low levels of IgG anti-γDPGAantibodies (Table 1). TABLE 1 Composition and serum geometric mean IgGanti-γDPGA and anti-carrier protein antibodies elicited in mice byconjugates of γPGA with BSA, rEPA and rPA. Mol γDPGA Protein per Anti-γDPGA* Anti-protein^(†) per mol γDPGA Second Third Second ThirdConjugate protein (wt/wt) injection injection injection injectionγDPGA-B. anthracis  NA^(‡) NA 0.3 4.4 NA NA rEPA-AH/γDPGA-B. anthracisNA 1:0.29 695 2312  ND^(§) ND rPA-AH γDPGA-B. anthracis NA 1:4.42 13253108 ND ND BSA-SH/Gly₃-γDPGA₁₀-C^(¶) 7 1:0.14 134 1984 ND NDBSA-SH/Gly₃-γDPGA₁₀-C 18 1:0.35 1882 1821 ND ND BSA-SH/Gly₃-γDPGA₁₀-C 251:0.49 2063 2780 ND ND BSA-SH/Gly₃-γLPGA₁₀-C 7 1:0.14 261 618 ND NDrEPA/Cys-Gly₃-γDPGA₁₀-C 7 1:0.14 479 4470 ND ND rEPA-SH/Gly₃-γDPGA₅-C 171:0.17 502 1168 ND ND rEPA-SH/Gly3-γDPGA₁₀-C 9 1:0.18 931 3193 ND NDrEPA-SH/Gly3-γDPGA₂₀-C 5 1:0.19 749 2710 ND ND rPA/Cys-Gly₃-γDPGA₅-C 321:0.26 2454 4560 0.06 8.5 rPA/Cys-Gly₃-γDPGA₁₀-C 16 1:0.26 9091 112681.30 59.3 rPA/Cys-Gly₃-γDPGA₂₀-C 14 1:0.44 742 3142 0.01 4.5rPA/Cys-Gly₃-γDPGA₅-N 22 1:0.18 3149 3460 3.70 95.0rPA/Cys-Gly₃-γDPGA₁₀-N 21 1:0.33 5489 7516 0.10 2.2rPA/Cys-Gly₃-γDPGA₂₀-N 8 1:0.25 2630 5461 0.05 4.9 rPA-SH/Gly₃-γDPGA₅-C15 1:0.12 1813 3607 0.27 19.7 rPA-SH/Gly₃-γDPGA₁₀-C 11 1:0.18 10460 99070.50 102.0 rPA-SH/Gly₃-γDPGA₁₀-C 14 1:0.22 4378 7206 0.34 66.3rPA-SH/Gly₃-γDPGA₂₀-C 4 1:0.13 2655 4069 0.90 32.2 rPA-SH/Gly₃-γDPGA₂₀-C8 1:0.25 9672 7320 0.22 189.0 rPA/Cys-Gly₃-γLPGA₂₀-N 22 1:0.70 24 790.14 3.0 rPA/Cys-Gly₃-γLPGA₂₀-C 24 1:0.76 155 437 0.31 7.8BSA-AH/CHO-Gly₃-γDPGA₁₀-C 12 1:0.23 1476 3354 ND NDrEPA-AH/CHO-Gly₃-γDPGA₁₀-C 8 1:0.15 807 2099 1 14rPA-AH/CHO-Gly₃-γDPGA₁₀-C 22 1:0.34 ND ND ND NDBSA-CHO/AH-Gly₃-γDPGA₁₀-N 8 1:0.17 185 1139 ND ND rEPA-CHO/AH-γDPGA₁₅-N6 1:0.18 ND ND ND ND rPA-CHO/AH-γDPGA₁₅-N 5 1:0.12 ND ND ND NDrPA_(form)-CHO/AH-Gly₃-γDPGA₁₀-N 29 1:0.45 ND ND ND NDBSA-SL-AH/CHO-Gly₃-γDPGA₁₀-C 3 1:0.06 103 822 ND ND rEPA/Cys-γDPGA₁₅-NND ND ND ND ND ND rPA_(form)/Cys-Gly₃-γDPGA₁₀-N 15 1:0.23 ND ND ND ND*γDPGA from B. anthracis (strain A34), 2.5 μg as a conjugate used forinjection; antibodies by ELISA expressed as EU.^(†)Antibodies by ELISA expressed as μg Ab/ml.^(‡)Not applicable^(§)Not done^(¶)C or N refers to the free amino acid on the γPGA bound to theprotein.

A dose response of two γDPGA conjugates with rPA and rEPA as the carriershowed that rPA was a more effective carrier than rEPA (Table 2). Bothpeptides had 20 glutamic acid residues, and similar number of chains percarrier protein. The lowest dose (2.5 μg) of rPA-SH/Gly₃-γDPGA-Celicited the highest level of IgG anti-γDPGA antibodies (9, 133 EU,Table 2). The levels declined about half that at the 20 μg dose (Table2). rEPA-SH/Gly₃-γDPGA-C, in contrast, elicited similar levels at alldosages (Table 2). TABLE 2 Dose/immunogenicity relation of conjugatesprepared with 20-mers of γDPGA bound to rPA or rEPA. Mol Dose/ Anti-γDPGA/ Protein/ mice γDPGA mol γDPGA (μg 3^(rd) Conjugate protein(wt/wt) γDPGA) injection rPA-SH/Gly₃-γDPGA₂₀-C 8 1:0.25 2.5 9152 5 707010 3487 20 4901 rEPA-SH/Gly₃-γDPGA₂₀-C 6 1:0.23 2.5 1956 5 2393 10 263920 2834Five- to six-week old NIH general purpose mice (n = 10) injected s.c.with 0.1 ml of the conjugates two weeks apart and exsanguinated sevendays after the third injection. IgG anti-γDPGA was measured by ELISA andthe results expressed as the geometric mean (9,152 vs. 3,487, P = 0.003;9,152 vs. 4,901, P = 0.04; 9,152 vs. 1,956, P < 0.0001; 7,070 vs. 2,393,P < 0.0001).

The relationship between γDPGA conjugate dosage and immunogenicity wasfurther examined using a γDPGA-rPA conjugate (rPA/Cys-Gly₃-γDPGA₁₀-N,with 22 chains per carrier protein) at doses ranging from 2.5 μg to 0.31μg per mouse (with 20 μg per mouse for comparison). The optimal responseto γDPGA was at 1.25 μg per mouse (Table 3). The response to rPAincreased with a higher immunizing dose (Table 3). TABLE 3Dose/immunogenicity relation of conjugate prepared with 10-mer of γDPGAbound to rPA. Dose Anti- γDPGA Anti-rPA μg/mouse 2^(nd) injection 3^(rd)injection 2^(nd) injection 3^(rd) injection 20 — 3716 — 437 2.5 22315812 2 206 1.25 2314 6241 2 118 0.63 984 4943 0.6 37 0.31 493 3480 0.3 9

The effect of adjuvant on immunogenicity was studied using two γDPGA-rPAconjugates. Injection of the conjugate with aluminum hydroxide improvedsignificantly the immune response to rPA (Table 4). The anti-γDPGAlevels were not statistically different (Table 4). TABLE 4 Formulationeffect. Anti- γDPGA Anti-rPA Dose 2^(nd) in- 3^(rd) in- 2^(nd) in-3^(rd) in- Conjugate μg/mouse jection jection jection jectionrPA/Cys-Gly₃- 2.5 2231 5812 2 206 γDPGA₁₀-N 2.5 + al* 3527 6231 80  282rPA/Cys-Gly₃- 2.5 1041 2315 1 185 γDPGA₁₀-C 1 — 2880 — 61 1 + form** —2556 — 23 1 + al — 3975 — 258 1 + form/al — 3268 — 297*aluminum hydroxide (Alhydrogel)**formaldehyde treatment (Porro et al., J. Infect. Dis. 142: 716-24,1980; Nencioni et al., Infect. Immun. 59: 625-30, 1991).

Example 3 Serum IgG Anti-Carrier Protein Antibodies

This example demonstrates that conjugates of B. anthracis γDPGA elicitedIgG anti-carrier protein antibodies in addition to anti-γDPGAantibodies.

With few exceptions, both the length and number of γDPGA chains percarrier protein were related to the level of IgG anti-carrier proteinantibodies (Table 1). Conjugates prepared with γDPGA polypeptidescontaining 20 residues elicited low levels of carrier protein antibodies(Table 1). Conjugates prepared with either 5 or 10 glutamic acidresidues pre chain, and conjugates with ≦15 chains per carrier proteinelicited the highest levels of IgG carrier protein antibodies (Table 1).

Example 4 Opsonophagocytic Activity of Mouse Antisera

This example demonstrates that IgG anti-DPGA antibodies haveopsonophagocytic activity.

Sera from normal mice or those immunized with rEPA or rPA did not haveopsonophagocytic activity. However, in mice immunized withBSA-SH/Gly₃-γDPGA₁₀-C or BSA-SH/Gly₃-γDPGA₁₀-C there was a correlationbetween the level of IgG anti-γDPGA antibodies and opsonophagocytosis(r=0.7, P=0.03, Table 5). Addition of γDPGA from B. anthracis to theimmune sera showed a dose-related reduction of the opsonophagocytictiter of approximately 60%. TABLE 5 Opsonophagocytic activity and IgGanti-γDPGA antibodies (ELISA) elicited by BSA-SH/Gly₃-γDPGA₁₀-C.Reciprocal Sera IgG anti-γDPGA opsonophagocytic titer 1196G 407 Notdetected 1195C 1,147 640 1197B 3,975 2,560 1190H 3,330 2,560 1194D 3,2782,560 1193B 3,178 2,560 1194G 3,277 2,560 1191J 5,191 5,120Correlation coefficient between ELISA and reciprocal opsonophagocytictiter is 0.7, P = 0.03.

Example 5 Methods for Preparing Peptide and Protein Mimetics

This example describes methods for preparing peptide and proteinmimetics modified at the N-terminal amino group, the C-terminal carboxylgroup, and/or changing one or more of the amido linkages in the peptideto a non-amido linkage. It is understood that two or more suchmodifications can be coupled in one peptide or protein mimetic structure(for example, modification at the C-terminal carboxyl group andinclusion of a —CH2-carbamate linkage between two amino acids in thepeptide).

For N-terminal modifications, peptides typically are synthesized as thefree acid but, as noted above, can be readily prepared as the amide orester. One can also modify the amino and/or carboxy terminus of peptidecompounds to produce other compounds useful within the disclosure. Aminoterminus modifications include methylating (that is, —NHCH3 or—NH(CH3)2), acetylating, adding a carbobenzoyl group, or blocking theamino terminus with any blocking group containing a carboxylatefunctionality defined by RCOO—, where R is selected from the groupconsisting of naphthyl, acridinyl, steroidyl, and similar groups.Carboxy terminus modifications include replacing the free acid with acarboxamide group or forming a cyclic lactam at the carboxy terminus tointroduce structural constraints. Amino terminus modifications are asrecited above and include alkylating, acetylating, adding a carbobenzoylgroup, forming a succinimide group, and the like. The N-terminal aminogroup can then be reacted as follows: (A) to form an amide group of theformula RC(O)NH— where R is as defined above by reaction with an acidhalide (for example, RC(O)Cl) or acid anhydride. Typically, the reactioncan be conducted by contacting about equimolar or excess amounts (forexample, about 5 equivalents) of an acid halide to the peptide in aninert diluent (for example, dichloromethane) preferably containing anexcess (for example, about 10 equivalents) of a tertiary amine, such asdiisopropylethylamine, to scavenge the acid generated during reaction.Reaction conditions are otherwise conventional (for example, roomtemperature for 30 minutes). Alkylation of the terminal amino to providefor a lower alkyl N-substitution followed by reaction with an acidhalide as described above will provide for N-alkyl amide group of theformula RC(O)NR—. (B) to form a succinimide group by reaction withsuccinic anhydride. As before, an approximately equimolar amount or anexcess of succinic anhydride (for example, about 5 equivalents) can beemployed and the amino group is converted to the succinimide by methodswell known in the art including the use of an excess (for example, tenequivalents) of a tertiary amine such as diisopropylethylamine in asuitable inert solvent (for example, dichloromethane) (see, for example,U.S. Pat. No. 4,612,132). It is understood that the succinic group canbe substituted with, for example, C2-C6 alkyl or —SR substituents thatare prepared in a conventional manner to provide for substitutedsuccinimide at the N-terminus of the peptide. Such alkyl substituentsare prepared by reaction of a lower olefin (C2-C6) with maleic anhydridein the manner described by Wollenberg et al. (U.S. Pat. No. 4,612,132)and —SR substituents are prepared by reaction of RSH with maleicanhydride where R is as defined above. (C) to form abenzyloxycarbonyl-NH— or a substituted benzyloxycarbonyl-NH— group byreaction with approximately an equivalent amount or an excess of CBZ-Cl(that is, benzyloxycarbonyl chloride) or a substituted CBZ-Cl in asuitable inert diluent (for example, dichloromethane) preferablycontaining a tertiary amine to scavenge the acid generated during thereaction. (D) to form a sulfonamide group by reaction with an equivalentamount or an excess (for example, 5 equivalents) of R—S(O)2Cl in asuitable inert diluent dichloromethane) to convert the terminal amineinto a sulfonamide where R is as defined above. Preferably, the inertdiluent contains excess tertiary amine (for example, ten equivalents)such as diisopropylethylamine, to scavenge the acid generated duringreaction. Reaction conditions are otherwise conventional (for example,room temperature for 30 minutes). (E) to form a carbamate group byreaction with an equivalent amount or an excess (for example, 5equivalents) of R—OC(O)Cl or R—OC(O)OC6H4-p-NO2 in a suitable inertdiluent (for example, dichloromethane) to convert the terminal amineinto a carbamate where R is as defined above. Preferably, the inertdiluent contains an excess (for example, about 10 equivalents) of atertiary amine, such as diisopropylethylamine, to scavenge any acidgenerated during reaction. Reaction conditions are otherwiseconventional (for example, room temperature for 30 minutes). (F) to forma urea group by reaction with an equivalent amount or an excess (forexample, 5 equivalents) of R—N═C═O in a suitable inert diluent (forexample, dichloromethane) to convert the terminal amine into a urea(that is, RNHC(O)NH—) group where R is as defined above. Preferably, theinert diluent contains an excess (for example, about 10 equivalents) ofa tertiary amine, such as diisopropylethylamine. Reaction conditions areotherwise conventional (for example, room temperature for about 30minutes).

In preparing peptide mimetics wherein the C-terminal carboxyl group isreplaced by an ester (that is, —C(O)OR where R is as defined above),resins as used to prepare peptide acids are typically employed, and theside chain protected peptide is cleaved with base and the appropriatealcohol, for example, methanol. Side chain protecting groups are thenremoved in the usual fashion by treatment with hydrogen fluoride toobtain the desired ester.

In preparing peptide mimetics wherein the C-terminal carboxyl group isreplaced by the amide —C(O)NR3R4, a benzhydrylamine resin is used as thesolid support for peptide synthesis. Upon completion of the synthesis,hydrogen fluoride treatment to release the peptide from the supportresults directly in the free peptide amide (that is, the C-terminus is—C(O)NH2). Alternatively, use of the chloromethylated resin duringpeptide synthesis coupled with reaction with ammonia to cleave the sidechain protected peptide from the support yields the free peptide amideand reaction with an alkylamine or a dialkylamine yields a side chainprotected alkylamide or dialkylamide (that is, the C-terminus is—C(O)NRR1 where R and R1 are as defined above). Side chain protection isthen removed in the usual fashion by treatment with hydrogen fluoride togive the free amides, alkylamides, or dialkylamines.

In other embodiments of the disclosure, the C-terminal carboxyl group ora C-terminal ester of a biologically active peptide can be induced tocyclize by internal displacement of the —OH or the ester (—OR) of thecarboxyl group or ester respectively with the N-terminal amino group toform a cyclic peptide. For example, after synthesis and cleavage to givethe peptide acid, the free acid is converted to an activated ester by anappropriate carboxyl group activator such as dicyclohexylcarbodiimide insolution, for example, in methylene chloride (CH2Cl2), dimethylformamide mixtures. The cyclic peptide is then formed by internaldisplacement of the activated ester with the N-terminal amine. Internalcyclization as opposed to polymerization can be enhanced by use of verydilute solutions. Such methods are well known in the art.

One can cyclize active peptides for use within the disclosure, orincorporate a desamino or descarboxy residue at the termini of thepeptide, so that there is no terminal amino or carboxyl group, todecrease susceptibility to proteases, or to restrict the conformation ofthe peptide. C-terminal functional groups among peptide analogs andmimetics of the present disclosure include amide, amide lower alkyl,amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lowerester derivatives thereof, and the pharmaceutically acceptable saltsthereof.

Other methods for making peptide and protein derivatives and mimeticsfor use within the methods and compositions of the disclosure aredescribed in Hruby et al., (Biochem. J. 268:249-62, 1990). According tothese methods, biologically active peptides and proteins serve asstructural models for non-peptide mimetic compounds having similarbiological activity as the native peptide or protein. Those of skill inthe art recognize that a variety of techniques are available forconstructing compounds with the same or similar desired biologicalactivity as the lead peptide or protein compound, or that have morefavorable activity than the lead with respect a desired property such assolubility, stability, and susceptibility to hydrolysis and proteolysis(see, for example, Morgan and Gainor, Ann. Rep. Med. Chem. 24:243-52,1989). These techniques include, for example, replacing a peptidebackbone with a backbone composed of phosphonates, amidates, carbamates,sulfonamides, secondary amines, and/or N-methylamino acids.

Peptide and protein mimetics wherein one or more of the peptidyllinkages (—C(O)NH—) have been replaced by such linkages as a—CH2-carbamate linkage, a phosphonate linkage, a —CH2-sulfonamidelinkage, a urea linkage, a secondary amine (—CH2NH—) linkage, and analkylated peptidyl linkage (—C(O)NR6—where R6 is lower alkyl) areprepared, for example, during conventional peptide synthesis by merelysubstituting a suitably protected amino acid analogue for the amino acidreagent at the appropriate point during synthesis. Suitable reagentsinclude, for example, amino acid analogues wherein the carboxyl group ofthe amino acid has been replaced with a moiety suitable for forming oneof the above linkages. For example, if one desires to replace a —C(O)NR—linkage in the peptide with a —CH2-carbamate linkage (—CH2OC(O)NR—),then the carboxyl (—COOH) group of a suitably protected amino acid isfirst reduced to the —CH2OH group which is then converted byconventional methods to a —OC(O)Cl functionality or apara-nitrocarbonate —OC(O)O—C6H4-p-NO2 functionality. Reaction of eitherof such functional groups with the free amine or an alkylated amine onthe N-terminus of the partially fabricated peptide found on the solidsupport leads to the formation of a —CH2OC(O)NR— linkage. For a moredetailed description of the formation of such —CH2-carbamate linkages,see, for example, Cho et al., Science 261:1303-05, 1993.

Replacement of an amido linkage in an active peptide with a—CH2-sulfonamide linkage can be achieved by reducing the carboxyl(—COOH) group of a suitably protected amino acid to the —CH2OH group,and the hydroxyl group is then converted to a suitable leaving groupsuch as a tosyl group by conventional methods. Reaction of thederivative with, for example, thioacetic acid followed by hydrolysis andoxidative chlorination will provide for the —CH2-S(O)2Cl functionalgroup which replaces the carboxyl group of the otherwise suitablyprotected amino acid. Use of this suitably protected amino acid analoguein peptide synthesis provides for inclusion of an —CH2S(O)2NR— linkagethat replaces the amido linkage in the peptide thereby providing apeptide mimetic. For a more complete description on the conversion ofthe carboxyl group of the amino acid to a —CH2S(O)2Cl group, see, forexample, Weinstein and Boris, Chemistry & Biochemistry of Amino Acids,Peptides and Proteins, Vol. 7, pp. 267-357, Marcel Dekker, Inc., NewYork, 1983. Replacement of an amido linkage in an active peptide with aurea linkage can be achieved, for example, in the manner set forth inU.S. patent application Ser. No. 08/147,805.

Secondary amine linkages wherein a —CH2NH— linkage replaces the amidolinkage in the peptide can be prepared by employing, for example, asuitably protected dipeptide analogue wherein the carbonyl bond of theamindo linkage has been reduced to a CH2 group by conventional methods.For example, in the case of diglycine, reduction of the amide to theamine will yield after deprotection H2NCH2CH2NHCH2 COOH that is thenused in N-protected form in the next coupling reaction. The preparationof such analogues by reduction of the carbonyl group of the amidolinkage in the dipeptide is well known in the art.

The biologically active peptide and protein agents of the presentdisclosure can exist in a monomeric form with no disulfide bond formedwith the thiol groups of cysteine residue(s) that may be present in thesubject peptide or protein. Alternatively, an intermolecular disulfidebond between thiol groups of cysteines on two or more peptides orproteins can be produced to yield a multimeric (for example, dimeric,tetrameric or higher oligomeric) compound. Certain of such peptides andproteins can be cyclized or dimerized via displacement of the leavinggroup by the sulfur of a cysteine or homocysteine residue (see, forexample, Barker et al., J. Med. Chem. 35:2040-48, 1992 and Or et al., J.Org. Chem. 56:3146-49, 1991). Thus, one or more native cysteine residuescan be substituted with a homocysteine. Intramolecular or intermoleculardisulfide derivatives of active peptides and proteins provide analogs inwhich one of the sulfurs has been replaced by a CH2 group or otherisostere for sulfur. These analogs can be made via an intramolecular orintermolecular displacement, using methods known in the art.

Example 6 Delivery of γPGA Conjugates

This example demonstrates that delivery of γPGA conjugates can beenhanced by methods and agents that target selective transportmechanisms and promote endo- or transcytocis of macromoloecular drugs.

In this regard, the compositions and delivery methods of the disclosureoptionally incorporate a selective transport-enhancing agent thatfacilitates transport of one or more biologically active agents. Thesetransport-enhancing agents can be employed in a combinatorialformulation or coordinate administration protocol with one or more ofthe peptides, proteins, analogs and mimetics disclosed herein, tocoordinately enhance delivery of the biologically active agent(s) intotarget cells. Exemplary selective transport-enhancing agents for usewithin this aspect of the disclosure include, but are not limited to,glycosides, sugar-containing molecules, and binding agents such aslectin binding agents, which are known to interact specifically withepithelial transport barrier components (see, for example, Goldstein etal., Annu. Rev. Cell. Biol. 1:1-39, 1985). For example, specific“bioadhesive” ligands, including various plant and bacterial lectins,which bind to cell surface sugar moieties by receptor-mediatedinteractions can be employed as carriers or conjugated transportmediators for enhancing delivery of γPGA conjugates within thedisclosure. Certain bioadhesive ligands for use within the disclosurewill mediate transmission of biological signals to epithelial targetcells that trigger selective uptake of the adhesive ligand byspecialized cellular transport processes (endocytosis or transcytosis).These transport mediators can therefore be employed as a “carriersystem” to stimulate or direct selective uptake of a γPGA conjugatewithin the methods of the disclosure. To utilize thesetransport-enhancing agents, general carrier formulation and/orconjugation methods known in the art are used to complex or otherwisecoordinately administer a selective transport enhancer (for example, areceptor-specific ligand) and a γPGA conjugate to trigger or mediateenhanced endo- or transcytosis of the γPGA conjugate into specifictarget cell(s), tissue(s) or compartment(s).

Lectins are plant proteins that bind to specific sugars found on thesurface of glycoproteins and glycolipids of eukaryotic cells.Concentrated solutions of lectins have a “mucotractive” effect, andvarious studies have demonstrated rapid receptor mediated endocytosis oflectins and lectin conjugates (for example, concanavalin A conjugatedwith colloidal gold particles) across mucosal surfaces. Additionalstudies have reported that the uptake mechanisms for lectins can beutilized for intestinal drug targeting in vivo. In certain of thesestudies, polystyrene nanoparticles (500 nm) were covalently coupled totomato lectin and reported yielded improved systemic uptake after oraladministration to rats. In addition to plant lectins, microbial adhesionand invasion factors provide a rich source of candidates for use asadhesive/selective transport carriers within the compositions andmethods of the disclosure (see, for example, Lehr, Crit. Rev. Therap.Drug Carrier Syst. 11:177-218, 1995 and Swann, Pharmaceutical Research15:826-32, 1998). Two components are necessary for bacterial adherenceprocesses, a bacterial “adhesin” (adherence or colonization factor) anda receptor on the host cell surface. Bacteria causing mucosal infectionsneed to penetrate the mucus layer before attaching themselves to theepithelial surface. This attachment is usually mediated by bacterialfimbriae or pilus structures, although other cell surface components canalso take part in the process. Adherent bacteria colonize mucosalepithelia by multiplication and initiation of a series of biochemicalreactions inside the target cell through signal transduction mechanisms(with or without the help of toxins).

Associated with these invasive mechanisms, a wide diversity ofbioadhesive proteins (for example, invasin, internalin) originallyproduced by various bacteria and viruses are known. These allow forextracellular attachment of such microorganisms with an impressiveselectivity for host species and even particular target tissues. Signalstransmitted by such receptor-ligand interactions trigger the transportof intact, living microorganisms into, and eventually through,epithelial cells by endo- and transcytotic processes. Such naturallyoccurring phenomena can be harnessed (for example, by complexing a γPGAconjugate with an adhesin) according to the teachings herein forenhanced delivery of γPGA conjugates and/or other biologically activecompounds. One advantage of this strategy is that the selective carrierpartners thus employed are substrate-specific, leaving the naturalbarrier function of epithelial tissues intact against other solutes(see, for example, Lehr, Drug Absorption Enhancement, pp. 325-362, deBoer, Ed., Harwood Academic Publishers, 1994).

Various bacterial and plant toxins that bind epithelial surfaces in aspecific, lectin-like manner are also useful within the methods andcompositions of the disclosure. For example, diphtheria toxin entershost cells rapidly by receptor mediated endocytosis. Likewise, the Bsubunit of the E. coli heat labile toxin binds to the brush border ofintestinal epithelial cells in a highly specific, lectin-like manner.Uptake of this toxin and transcytosis to the basolateral side of theenterocytes has been reported in vivo and in vitro. Other researcheshave expressed the transmembrane domain of diphtheria toxin in E. colias a maltose-binding fusion protein and coupled it chemically to high-Mwpoly-L-lysine. The resulting complex was successfully used to mediateinternalization of a reporter gene in vitro. In addition to theseexamples, Staphylococcus aureus produces a set of proteins (for example,staphylococcal enterotoxin A, staphylococcal enterotoxin B and toxicshock syndrome toxin 1) which act both as superantigens and toxins.Studies relating to these proteins have reported dose-dependent,facilitated transcytosis of staphylococcal enterotoxin B and toxic shocksyndrome toxin 1 in Caco-2 cells.

Various plant toxins, mostly ribosome-inactivating proteins, have beenidentified that bind to any mammalian cell surface expressing galactoseunits and are subsequently internalized by receptor mediatedendocytosis. Toxins such as nigrin b, sarcin, ricin and saporin,viscumin, and modeccin are highly toxic upon oral administration (thatis, they are rapidly internalized). Therefore, modified, less toxicsubunits of these compound will be useful within the disclosure tofacilitate the uptake of γPGA conjugates and other biologically activeagents, including PA, other bacterial products and analogs, variants,derivatives and mimetics thereof.

Viral hemagglutinins include another type of transport agent tofacilitate delivery of γPGA conjugates and other biologically activeagents within the methods and compositions of the disclosure. Theinitial step in many viral infections is the binding of surface proteins(hemagglutinins) to mucosal cells. These binding proteins have beenidentified for most viruses, including rotaviruses, Varicella zostervirus, semliki forest virus, adenoviruses, potato leafroll virus, andreovirus. These and other exemplary viral hemagglutinins can be employedin a combinatorial formulation (for example, a mixture or conjugateformulation) or coordinate administration protocol with, for example,one or more γPGA conjugates, PA immunogens, other bacterial products, oranalogs, variants, derivatives and mimetics thereof. Alternatively,viral hemagglutinins can be employed in a combinatorial formulation orcoordinate administration protocol to directly enhance delivery of aγPGA conjugate or other biologically active agent within the disclosure.

A variety of endogenous, selective transport-mediating factors are alsoavailable for use within the disclosure. Exemplary among these areprotocytotic transport carriers within the folate carrier system, whichmediate transport of the vitamin folic acid into target cells viaspecific binding to the folate receptor (see, for example, Reddy et al.,Crit. Rev. Ther. Drug Car. Syst. 15:587-27, 1998). This receptor systemhas been used in drug-targeting approaches to cancer cells, but also inprotein delivery, gene delivery, and targeting of antisenseoligonucleotides to a variety of cell types. Folate-drug conjugates arewell suited for use within the methods and compositions of thedisclosure, because they allow penetration of target cells exclusivelyvia folate receptor-mediated endocytosis. When folic acid is covalentlylinked to a biologically active agent, folate receptor binding affinity(KD˜10-10M) is not significantly compromised, and endocytosis proceedsrelatively unhindered, promoting uptake of the attached active agent bythe folate receptor-expressing cell.

In addition to the folate receptor pathway, a variety of additionalmethods to stimulate transcytosis within the disclosure are directed tothe transferrin receptor pathway, and the riboflavin receptor pathway.In one aspect, conjugation of a PGA conjugate or other biologicallyactive agent to riboflavin can effectuate receptor mediated endocytosisuptake. Yet additional embodiments of the disclosure utilize vitamin B12(cobalamin) as a specialized transport protein (for example, conjugationpartner) to facilitate entry of γPGA conjugates and other biologicallyactive agents into target cells. Certain studies suggest that thisparticular system can be employed for mucosal delivery into theintestine. Still other embodiments of the disclosure utilize transferrinas a carrier or stimulant of receptor mediated endocytosis of mucosallydelivered biologically active agents. Transferrin, an 80 kDairon-transporting glycoprotein, is efficiently taken up into cells byreceptor mediated endocytosis. Transferrin receptors are found on thesurface of most proliferating cells, in elevated numbers onerythroblasts and on many kinds of tumors. Each of the foregoing agentsthat stimulate receptor-mediated transport can be employed within themethods of the disclosure as combinatorially formulated (for example,conjugated) and/or coordinately administered agents to enhancereceptor-mediated transport of γPGA conjugates and other biologicallyactive agents, including, PA, carriers, linkers, and other bacterialtoxins and analogs, variants, derivatives and mimetics thereof.

Immunoglobulin transport mechanisms provide yet additional endogenouspathways and reagents for enhancing delivery of γPGA conjugates andother active agents within the methods and compositions of thedisclosure. Receptor-mediated transcytosis of immunoglobulin G (IgG)across the neonatal small intestine serves to convey passive immunity tomany newborn mammals. Within the methods and compositions of the presentdisclosure, IgG and other immune system-related carriers (includingpolyclonal and monoclonal antibodies and various fragments thereof) canbe complexed or otherwise coordinately administered with γPGA conjugatesand other biologically active agents to provide for targeted delivery,typically by receptor-mediated transport. For example, the γPGAconjugate or other biologically active agent can be covalently linked tothe IgG or other immunological active agent or, alternatively,formulated in liposomes or other carrier vehicle which is in turnmodified (for example, coated or covalently linked) to incorporate IgGor other immunological transport enhancer. In certain embodiments,polymeric IgA and/or IgM transport agents are employed, which bind tothe polymeric immunoglobulin receptors of target epithelial cells.Within these methods, expression of polymeric immunoglobulin receptorscan be enhanced by cytokines.

Within more detailed aspects of the disclosure, antibodies and otherimmunological transport agents can be themselves modified for enhanceddelivery of γPGA conjugates or other biologically active agents. Forexample, antibodies can be more effectively administered within themethods and compositions of the disclosure by charge modifyingtechniques. In one such aspect, an antibody drug delivery strategyinvolving antibody cationization is utilized that facilitates bothtrans-endothelial migration and target cell endocytosis (see, forexample, Pardridge, et al., JPET 286:548-44, 1998). In one suchstrategy, the pI of the antibody is increased by converting surfacecarboxyl groups of the protein to extended primary amino groups. Thesecationized homologous proteins have no measurable tissue toxicity andhave minimal immunogenicity. In addition, monoclonal antibodies can becationized with retention of affinity for the target protein.

Additional selective transport-enhancing agents for use within thedisclosure include whole bacteria and viruses, including geneticallyengineered bacteria and viruses, as well as components of such bacteriaand viruses. This aspect of the disclosure includes the use of bacterialghosts and subunit constructs, for example, as described by Huter etal., J. Control. Rel. 61:51-63, 1999. Bacterial ghosts are non-denaturedbacterial cell envelopes, for example as produced by the controlledexpression of the plasmid-encoded lysis gene E of bacteriophage PhiX174in gram-negative bacteria. Protein E-specific lysis does not cause anyphysical or chemical denaturation to bacterial surface structures, andbacterial ghosts are therefore useful in development of inactivatedwhole-cell vaccines. Ghosts produced from Actinobacilluspleuropneumoniae, Pasteurella haemolytica and Salmonella sp. have provedsuccessful in vaccination experiments. Recombinant bacterial ghosts canbe created by the expression of foreign genes fused to amembrane-targeting sequence, and thus can carry foreign therapeuticpeptides and proteins anchored in their envelope. The fact thatbacterial ghosts preserve a native cell wall, including bioadhesivestructures like fimbriae of their living counterparts, makes themsuitable for the attachment to specific target tissues such as mucosalsurfaces. Bacterial ghosts have been shown to be readily taken up bymacrophages, thus adhesion of ghosts to specific tissues can be followedby uptake through phagocytes.

In view of the foregoing, a wide variety of ligands involved inreceptor-mediated transport mechanisms are known in the art and can bevariously employed within the methods and compositions of the disclosure(for example, as conjugate partners or coordinately administereddelivery enhancers) to enhance delivery or receptor-mediated transportof γPGA conjugates and other biologically active agents, including PA orother bacterial products. Generally, these ligands include hormones andgrowth factors, bacterial adhesins and toxins, lectins, metal ions andtheir carriers, vitamins, immunoglobulins, whole viruses and bacteria orselected components thereof. Exemplary ligands among these classesinclude, for example, calcitonin, prolactin, epidermal growth factor,glucagon, growth hormone, estrogen, lutenizing hormone, platelet derivedgrowth factor, thyroid stimulating hormone, thyroid hormone, choleratoxin, diphtheria toxin, E. coli heat labile toxin, Staphylococcalenterotoxins A and B, ricin, saporin, modeccin, nigrin, sarcin,concanavalin A, transcobalantin, catecholamines, transferrin, folate,riboflavin, vitamin B1, low density lipoprotein, maternal IgO, polymericIgA, adenovirus, vesicular stomatitis virus, Rous sarcoma virus, V.cholerae, Kiebsiella strains, Serratia strains, parainfluenza virus,respiratory syncytial virus, Varicella zoster, and Enterobacter strains(see, for example, Swann, Pharmaceutical Research 15:826-32, 1998).

In certain additional embodiments of the disclosure, membrane-permeablepeptides (for example, “arginine rich peptides”) are employed tofacilitate delivery of γPGA conjugates or other biologically activeagents of the disclosure. While the mechanism of action of thesepeptides remains to be fully elucidated, they provide useful deliveryenhancing adjuncts for use within the compositions and methods herein.In one example, a basic peptide derived from human immunodeficiencyvirus (HIV)-1 Tat protein (for example, residues 48-60) facilitatestranslocation through cell membranes and can be utilized for enhancingdelivery of exogenous proteins and peptides into cells. The sequence ofTat (GRKKRRQRRRPPQ, SEQ ID NO: 1) includes a highly basic andhydrophilic peptide, which contains 6 arginine and 2 lysine residues inits 13 amino acid residues. Various other arginine-rich peptides havebeen identified which have a translocation activity similar toTat-(48-60). These include such peptides as the D-amino acid- andarginine-substituted Tat-(48-60), the RNA-binding peptides derived fromvirus proteins, such as HIV-1 Rev, and flock house virus coat proteins,and the DNA binding segments of leucine zipper proteins, such ascancer-related proteins c-Fos and c-Jun, and the yeast transcriptionfactor GCN4 (see, for example, Futaki et al., J. Biol. Chem.276:5836-40, 2000).

While this disclosure has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations of the preferred embodiments may be used and it isintended that the disclosure may be practiced otherwise than asspecifically described herein. Accordingly, this disclosure includes allmodifications encompassed within the spirit and scope of the disclosureas defined by the claims below.

1. An immunogenic conjugate comprising a a synthetic homopolymerpolypeptide of poly-γ-glutamic acid (γPGA) polypeptide covalently linkedto a carrier, wherein the conjugate elicits an immune response in asubject.
 2. The conjugate of claim 1, wherein the conjugate comprises aγPGA polypeptide comprising 5-20 glutamic acid residues.
 3. Theconjugate of claim 1, wherein the conjugate comprises a γPGA polypeptidecomprising 10-15 glutamic acid residues.
 4. The conjugate of claim 1,wherein the conjugate comprises a decameric γPGA polypeptide.
 5. Theconjugate of claim 1, wherein the carrier is selected from the groupconsisting of: (a) bovine serum albumin, (b) recombinant B. anthracisprotective antigen, (c) recombinant P. aeruginosa exotoxin A, (d)tetanus toxoid, (e) diphtheria toxoid, (f) pertussis toxoid, (g) C.perfringens toxoid, (h) hepatitis B surface antigen, (i) hepatitis Bcore antigen, (j) keyhole limpet hemocyanin, (k) horseshoe crabhemocyanin, (l) edestin, (m) mammalian serum albumins, (n) mammalianimmunoglobulins, analogs or mimetics of (a)-(n), and combinations of twoor more thereof.
 6. The conjugate of claim 1, wherein the carriercomprises recombinant B. anthracis protective antigen.
 7. (canceled) 8.The conjugate of claim 1, wherein the poly-γ-glutamic acid (γPGA)polypeptide comprises the D- or L-conformation.
 9. The conjugate ofclaim 1, wherein the poly-γ-glutamic acid (γPGA) polypeptide comprises aγDPGA polypeptide.
 10. The conjugate of claim 1, wherein thepoly-γ-glutamic acid (γPGA) polypeptide comprises a decameric γDPGApolypeptide and the carrier comprises recombinant B. anthracisprotective antigen.
 11. The conjugate of claim 1, wherein the carrier iscovalently linked to either the amino or carboxyl terminus of thepoly-γ-glutamic acid (γPGA) polypeptide.
 12. The conjugate of claim 1,wherein the carrier is covalently linked to the poly-γ-glutamic acid(γPGA) polypeptide via a thioether, disulfide, or amide bond.
 13. Theconjugate of claim 1, wherein the density of poly-γ-glutamic acid (γPGA)polypeptide to carrier is between about 5:1 and about 32:1.
 14. Theconjugate of claim 1, wherein the density of poly-γ-glutamic acid (γPGA)polypeptide to carrier is between about 10:1 and about 15:1.
 15. Theconjugate of claim 1, wherein the γPGA polypeptide is covalently linkedto the carrier via an aldehyde (CHO)/adipic acid hydrazide (AH) linkage.16. A composition comprising the conjugate of claim 1 and apharmaceutically acceptable carrier.
 17. The composition of claim 16,further comprising an adjuvant.
 18. A composition comprising theconjugate of claim 9 and a pharmaceutically acceptable carrier.
 19. Thecomposition of claim 18, further comprising an adjuvant.
 20. A method ofeliciting an immune response against a Bacillus antigenic epitope in asubject, comprising introducing into the subject the composition ofclaim 17, thereby eliciting an immune response in the subject.
 21. Themethod of claim 20, wherein the immune response is elicited against theBacillus capsular poly-γ-glutamic acid (γPGA) polypeptide.
 22. Themethod of claim 20, wherein the immune response is elicited against theBacillus capsular poly-γ-glutamic acid (γPGA) polypeptide and thecarrier. 23-33. (canceled)
 34. An immunogenic conjugate comprising aBacillus capsular poly-γ-glutamic acid (γPGA) polypeptide covalentlylinked to a carrier, wherein the carrier is selected from the groupconsisting of: (a) recombinant B. anthracis protective antigen, (b)recombinant P. aeruginosa exotoxin A, (c) tetanus toxoid, (d) diphtheriatoxoid, (e) pertussis toxoid, (f) C. perfringens toxoid, (g) hepatitis Bsurface antigen, (h) hepatitis B core antigen, (i) keyhole limpethemocyanin, (j) horseshoe crab hemocyanin, (k) edestin, (l) mammalianserum albumins, analogs or mimetics of (a)-(l), and combinationsthereof, and wherein the conjugate elicits an immune response in asubject.
 35. The conjugate of claim 34, wherein the carrier comprisesrecombinant B. anthracis protective antigen.
 36. The conjugate of claim34, wherein the Bacillus capsular γPGA polypeptide comprises a B.anthracis, B. licheniformis, B. pumilus, or B. subtilis γPGApolypeptide.
 37. The conjugate of claim 34, wherein the Bacilluscapsular γPGA polypeptide comprises the D- or L-conformation.
 38. Theconjugate of claim 34, wherein the Bacillus capsular γPGA polypeptidecomprises a γDPGA polypeptide.
 39. The conjugate of claim 34, whereinthe carrier is covalently linked to either the amino or carboxylterminus of the Bacillus capsular γPGA polypeptide.
 40. The conjugate ofclaim 34, wherein the carrier is covalently linked to the Bacilluscapsular γPGA polypeptide via a thioether, disulfide, or amide bond. 41.The conjugate of claim 34, wherein the Bacillus capsular γPGApolypeptide is covalently linked to the carrier via an aldehyde(CHO)/adipic acid hydrazide (AH) linkage.
 42. A composition comprisingthe conjugate of claim 34 and a pharmaceutically acceptable carrier. 43.The composition of claim 42, further comprising an adjuvant.
 44. Amethod of eliciting an immune response against a Bacillus antigenicepitope in a subject, comprising introducing into the subject thecomposition of claim 43, thereby eliciting an immune response in thesubject.
 45. The method of claim 44, wherein the immune response iselicited against the Bacillus capsular poly-γ-glutamic acid (γPGA)polypeptide.
 46. The method of claim 44, wherein the immune response iselicited against the Bacillus capsular poly-γ-glutamic acid (γPGA)polypeptide and the carrier.